[Discussion on the indications of internal mammary sentinel lymph node biopsy in breast cancer in the era of precision medicine].

Objective: To determine the clinical benefits of internal mammary sentinel lymph node biopsy (IM-SLNB) acquired by breast cancer patients with clinically positive axillary lymph node (ALN), and further optimize the IM-SLNB indications. Methods: All primary breast cancer patients with clinically positive ALN from February 2014 to September 2017 were prospectively recruited in this study. IM-SLNB was performed under the guidance of the modified injection technique. The success rate and visualization rate of IM-SLNB, metastatic rate of internal mammary sentinel lymph node (IMSLN) and its related factors were analyzed, and the clinical benefits were accessed according to the current guidelines. Results: Among 126 patients, all of 94 patients (74.6%) who showed internal mammary drainage successfully underwent IM-SLNB. The incidence of internal mammary artery bleeding and pleural lesion were 4.3%(4/94) and 9.6%(9/94), respectively. The metastatic rate of IMSLN was 38.3% (36/94), which was significantly associated with the number of positive ALN (P<0.001) and tumor size (P=0.024). The lymph node staging of 94 patients who underwent IM-SLNB was more accurate. Among them, 36 cases with positive IMSLN underwent internal mammary radiotherapy (IMRT), while the other 58 cases with negative IMSLN avoided radiotherapy. Conclusions: IM-SLNB should be routinely performed in patients with positive ALN. IM-SLNB can provide more accurate staging and guide tailored IMRT to benefit more breast cancer patients.

[Efficiency of the Modified Ottawa ankle rules for the differential diagnosis of fracture in acute foot and ankle injury].

Objective: To evaluate the efficiency of modified Ottawa Ankle Rules (OAR) for the differential diagnosis of fractures in acute foot and ankle injuries. Methods: From October 2016 to December 2016, 272cases (135 males and 137 females) of foot and ankle injury in emergency department of Tianjin Hospital were prospective enrolled in the study.The median age was 27.5 years (7-87); left limb 155, right 117 cases; injury time ranged from 0.3 to 24 h (median 4 h). Conventional and modified OAR was applied on physical examination, subsequently radiography performed to determine the occurrence of fractures.The efficiency of the two methods were compared and analyzed. Results: Fractures were found in 100 cases (36.8%), 49 cases of ankle and 51 cases of foot fractures.With the imaging results as the standard, the sensitivity for conventional and modified OAR were 93.0% and 100%, specificity were 9.9% and 8.7%, the positive predictive value were 37.5% and 38.9%, the negative predictive value were 70.8% and 100%, the accuracy were 40.4% and 42.3%, missed diagnosis rate were 7% and 0% respectively.The sensitivity, positive likelihood ratio, positive predictive value, negative predictive value, accuracy, negative likelihood ratio and missed diagnosis ratio were better than in modified OAR compared with Conventional OAR, while the specificity was slightly lower compared to Conventional OAR.The Kappa value of modified OAR was 0.065 (P>0.05), which is better than conventional OAR.Conventional OAR can reduce 6.3% (17/272) X-ray and modified OAR decline 5.5% (15/272). Conclusion: Modified OAR significantly reduces the rate of missed diagnosis of foot fractures, but its specificity is poor. Ultrasound can be assisted to improve the specificity and reduce the number of unnecessary X-rays.

Consumption of nuts and legumes and risk of stroke: a meta-analysis of prospective cohort studies.

BACKGROUND AND AIM: The relationships between dietary nuts and legume intake and risk of stroke are inconsistent. We summarized the evidence by a meta-analysis of prospective cohort studies. METHODS AND RESULTS: We systematically searched the MEDLINE and EMBASE databases up to 31 January 2014. Random-effects models were used to calculate summary relative risks (SRRs) and 95% confidence intervals (CIs). Between-study heterogeneity was assessed using the Cochran's Q and I(2) statistics. Eight prospective studies with a total of 468,887 subjects and 10,493 stroke events were included in the meta-analysis. Overall, a diet containing greater amounts of legumes may be not associated with a lower risk of stroke (SRR = 0.95, 95% CI: 0.84-1.08; P(heterogeneity) = 0.091, I(2) = 43.2%); however, a diet containing greater amounts of nuts may be associated with a lower risk of stroke (SRR = 0.90, 95% CI: 0.81-0.99; P(heterogeneity) = 0.527, I(2) = 0). Gender significantly modified the effects of nut consumption on stroke risk, and high nut intake was associated with reduced risk of stroke in women (SRR = 0.85, 95% CI: 0.75-0.97) other than in men (SRR = 0.95, 95% CI: 0.82-1.11). CONCLUSION: The current meta-analysis provides some evidences for the hypothesis that high intake of dietary nut was inversely associated with stroke risk, whereas dietary legumes intake was not associated with stroke risk.

Preparation, characterisation and evaluation of curcumin with piperine-loaded cubosome nanoparticles.

OBJECTIVE: In this study, curcumin was designed into the nanoformulation called cubosome with piperine in order to improve oral bioavailability and tissue distribution of curcumin. METHODS: The characteristic of the cubosome was studied by using scanning electron microscope (SEM), Infrared spectrum and small angle X-ray scattering (SAXS) techniques. Tissue distribution of cubosome was measured by liquid chromatography-mass spectrometry (LC-MS) method in mice. RESULTS: The characteristic of the cubosome was demonstrated that the curcumin and piperine were encapsulated in the interior of the cubosome and the crystal form was Pn3m space. The pharmacokinetic test revealed that the cubosome could improve the oral bioavailability significantly compared to the suspension of curcumin with piperine and be mainly absorbed by the spleen. CONCLUSION: These findings provide the reference to a preferable choice of the curcumin formulation and contribute to therapeutic application in clinical research.

Dendritic cell TLR4 induces Th1-type immune response against Cryptosporidium parvum infection.

The objective of this study was to investigate the mechanism of Toll-like receptor (TLR4)- mediated dendritic cell (DC) immune against Cryptosporidium parvum infection. C. parvum sporozoites were labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester. Murine bone marrow-derived DCs were isolated, and divided into TLR4 antibody blocking (TAB; infected with 2 × 105 labeled sporozoites and 0.5 µg TLR4 blocking antibody), TLR4 antibody unblocking (TAU; infected with 2 × 105 labeled sporozoites), and blank control (BC; with 1.5 mL Roswell Park Memorial Institute 1640 medium) groups. The adhesion of Cryptosporidium sporozoites to DCs and CD11c+ levels were examined by fluorescence microscopy and flow cytometry. Male KM mice were orally injected with C. parvum. The proliferation of T lymphocytes in spleen, expression of cytokines in peripheral blood, and TLR4 distribution features in different organs were further determined by immunohistochemistry. A significantly higher expression of CD11c+ and higher C. parvum sporozoite adhesion were found in the TAU group compared with other groups. The expression of CD4+CD8- /CD8+CD4- in the spleen were obviously differences between the TAB and TAU groups. The expression of TLR4, interleukin IL-4, IL-12, IL-18 and IFN-γ improved in the TAU group compared with TAB group. Higher expression of TLR4 was detected in the lymph nodes of mice in the TAU group, with pathological changes in the small intestine. Hence, TLR4 could mediate DCs to recognize C. parvum, inducing Th1 immune reaction to control C. parvum infection.

Inhibition of fengycins on the production of fumonisin B1 from Fusarium verticillioides.

AIM: To understand the role of fengycins in regulating the fumonisin B1 (FB1)production of Fusarium verticillioides. METHODS AND RESULTS: The mass ratio of FB1 to mycelia was determined in order to identify the effect of fengycins on FB1 production. It was shown that the amount of FB1 produced by unit mass mycelia decreased to 28% of the control. Results from mycelia resuspension with fengycins also demonstrated that fengycins had a potent impact on FB1 production. Gene expression patterns using quantitative reverse-transcription PCR (RT-PCR) revealed that the transcriptional levels of both FUM1 and FUM8 (coding enzymes for the generation of FB1) were down-regulated with fengycin treatment. CONCLUSIONS: Fengycins could down-regulate the transcription of some key genes involved in the production of FB1, and impair FB1 synthesis by F. verticillioides. SIGNIFICANCE AND IMPACT OF THE STUDY: These results further improved our understanding of fengycins as the potential candidates to control FB1 contamination in crops and food.

Importance of peripheral insulin levels for insulin-induced suppression of glucose production in depancreatized dogs.

It is generally believed that glucose production (GP) cannot be adequately suppressed in insulin-treated diabetes because the portal-peripheral insulin gradient is absent. To determine whether suppression of GP in diabetes depends on portal insulin levels, we performed 3-h glucose and specific activity clamps in moderately hyperglycemic (10 mM) depancreatized dogs, using three protocols: (a) 54 pmol.kg-1 bolus + 5.4 pmol.kg-1.min-1 portal insulin infusion (n = 7; peripheral insulin = 170 +/- 51 pM); (b) an equimolar peripheral infusion (n = 7; peripheral insulin = 294 +/- 28 pM, P < 0.001); and (c) a half-dose peripheral infusion (n = 7), which gave comparable (157 +/- 13 pM) insulinemia to that seen in protocol 1. Glucose production, use (GU) and cycling (GC) were measured using HPLC-purified 6-[3H]- and 2-[3H]glucose. Consistent with the higher peripheral insulinemia, peripheral infusion was more effective than equimolar portal infusion in increasing GU. Unexpectedly, it was also more potent in suppressing GP (73 +/- 7 vs. 55 +/- 7% suppression between 120 and 180 min, P < 0.001). At matched peripheral insulinemia (protocols 2 and 3), not only stimulation of GU, but also suppression of GP was the same (55 +/- 7 vs. 63 +/- 4%). In the diabetic dogs at 10 mM glucose, GC was threefold higher than normal but failed to decrease with insulin infusion by either route. Glycerol, alanine, FFA, and glucagon levels decreased proportionally to peripheral insulinemia. However, the decrease in glucagon was not significantly greater in protocol 2 than in 1 or 3. When we combined all protocols, we found a correlation between the decrements in glycerol and FFAs and the decrease in GP (r = 0.6, P < 0.01). In conclusion, when suprabasal insulin levels in the physiological postprandial range are provided to moderately hyperglycemic depancreatized dogs, suppression of GP appears to be more dependent on peripheral than portal insulin concentrations and may be mainly mediated by limitation of the flow of precursors and energy substrates for gluconeogenesis and by the suppressive effect of insulin on glucagon secretion. These results suggest that a portal-peripheral insulin gradient might not be necessary to effectively suppress postprandial GP in insulin-treated diabetics.

Proteolytic disruption of laminin-integrin complexes on muscle cells during synapse formation.

To explore whether a neural modulation of muscle integrins' extracellular ligand interactions contributes to synapse induction, we compared the distributions of beta1-integrins and basal lamina proteins on Xenopus myotomal myocytes developing in culture. beta1-Integrins formed numerous organized aggregates scattered over the entire muscle surface, with particularly dense accumulations at specialized sites resembling myotendinous and neuromuscular junctions. Integrin aggregates on muscle cells differed from those on surrounding fibroblasts and epithelial cells, both in their lack of response to cross-linking by multivalent ligands and in their consistent association with the cells' own extracellular matrices. Muscle integrin clusters were usually associated with congruent basal lamina accumulations containing laminin and a heparan sulfate proteoglycan (HSPG), sometimes including fibronectin and vitronectin acquired from the surrounding medium. Immediately prior to synaptic differentiation, any existing laminin and HSPG accumulations along the path of cell contact were eliminated, disrupting otherwise stable laminin-integrin complexes. This apparently proteolytic modulation of integrins' extracellular ligand interactions was soon followed by the accumulation of new congruent accumulations of laminin and HSPG in the developing synaptic basal lamina. Combining these results with earlier findings, we consider the possibility that postsynaptic differentiation is induced, at least in part, by the proteolytic disruption of integrin-ligand complexes at sites of nerve-muscle contact.

Molecular mechanism for the Shp-2 tyrosine phosphatase function in promoting growth factor stimulation of Erk activity.

We have previously shown that activation of extracellular signal-regulated kinase (Erk) by epidermal growth factor (EGF) treatment was significantly decreased in mouse fibroblast cells expressing a mutant Shp-2 molecule lacking 65 amino acids in the SH2-N domain, Shp-2(Delta46-110). To address the molecular mechanism for the positive role of Shp-2 in mediating Erk induction, we evaluated the activation of signaling components upstream of Erk in Shp-2 mutant cells. EGF-stimulated Ras, Raf, and Mek activation was significantly attenuated in Shp-2 mutant cells, suggesting that Shp-2 acts to promote Ras activation or to suppress the down-regulation of activated Ras. Biochemical analyses indicate that upon EGF stimulation, Shp-2 is recruited into a multiprotein complex assembled on the Gab1 docking molecule and that Shp-2 seems to exert its biological function by specifically dephosphorylating an unidentified molecule of 90 kDa in the complex. The mutant Shp-2(Delta46-110) molecule failed to participate in the Gab1-organized complex for dephosphorylation of p90, correlating with a defective activation of the Ras-Raf-Mek-Erk cascade in EGF-treated Shp-2 mutant cells. Evidence is also presented that Shp-2 does not appear to modulate the signal relay from EGF receptor to Ras through the Shc, Grb2, and Sos proteins. These results begin to elucidate the mechanism of Shp-2 function downstream of a receptor tyrosine kinase to promote the activation of the Ras-Erk pathway, with potential therapeutic applications in cancer treatment.

A deletion mutation in the SH2-N domain of Shp-2 severely suppresses hematopoietic cell development.

Shp-1 and Shp-2 are cytoplasmic protein tyrosine phosphatases that contain two Src homology 2 (SH2) domains. A negative regulatory role of Shp-1 in hematopoiesis has been strongly implicated by the phenotype of motheaten mice with a mutation in the Shp-1 locus, which is characterized by leukocyte hypersensitivity, deregulated mast cell function, and excessive erythropoiesis. A targeted deletion of 65 amino acids in the N-terminal SH2 (SH2-N) domain of Shp-2 leads to an embryonic lethality at midgestation in homozygous mutant mice. To further dissect the Shp-2 function in hematopoietic development, we have isolated homozygous Shp-2 mutant embryonic stem (ES) cells. Significantly reduced hematopoietic activity was observed when the mutant ES cells were allowed to differentiate into embryoid bodies (EBs), compared to the wild-type and heterozygous ES cells. Further analysis of ES cell differentiation in vitro showed that mutation in the Shp-2 locus severely suppressed the development of primitive and definitive erythroid progenitors and completely blocked the production of progenitor cells for granulocytes-macrophages and mast cells. Reverse transcriptase PCR analysis of the mutant EBs revealed reduced expression of several specific marker genes that are induced during blood cell differentiation. Stem cell factor induction of mitogen-activated protein kinase activity was also blocked in Shp-2 mutant cells. Taken together, these results indicate that Shp-2 is an essential component and primarily plays a positive role in signaling pathways that mediate hematopoiesis in mammals. Furthermore, stimulation of its catalytic activity is not sufficient, while interaction via the SH2 domains with the targets or regulators is necessary for its biological functions in cells. The in vitro ES cell differentiation assay can be used as a biological tool in dissecting cytoplasmic signaling pathways.

[A childhood-onset rapid-onset dystonia parkinsonism family with ATP1A3 gene mutation and literatures review].

Objective: To explore clinical characteristics, treatment, and prognosis of a family with childhood-onset rapid-onset dystonia parkinsonism (RDP) caused by ATP1A3 gene mutation and review literatures. Method: The clinical data of a RDP child, his brother and mother had been analyzed retrospectively. This family was admitted to Xiangya Hospital in January 2016. DNA samples were analyzed by the next-generation sequencing and confirmed by Sanger sequencing. Related literature from PubMed, Online Mendelian Inheritance in Man (OMIM), CNKI and Wanfang databases to date (up to October 2016) with"Rapid-onset dystonia-parkinsonism""RDP""DYT12" as key words was reviewed. Result: The proband boy was three years and four months old (April 2015) when he had the first attack of the disease. After a febricity, he suddenly acquired acute aphasia and limb movement disorder. Rehabilitation therapy and supportive treatment made his speech gradually recovered but still slurred. However, his abnormal walking posture still existed. Nine months later (January 2016, 4 years and one months old), symptoms including aphasia, dysphagia, and weakness with rostrocaudal gradient reoccured after fever. The disease progressed to the critical condition within 24 hours. He"seizured" four times with tonic spasms of limbs but without loss of consciousness. Family history showed his grandparents were consanguineous marriage. His mother and brother also developed abnormal gait and dysarthria after an infection before primary school age. Their symptoms improved gradually without relapsing. However, they did not recover entirely with mild intellectual disability. His mother had a healthy brother and sister. This proband had no other siblings but the brother. Heterozygous missense mutation p. R756H in ATP1A3 gene was detected in this proband, his mother and his brother. This mutation had been reported pathogenically related to RDP, and it located in highly conserved gene region. Benzodiazepine was used for the proband and his brother, with the proband being improved better although not completely. Meanwhile, benzodiazepine had no significant effect on his mother because of poor compliance. This is the first case report of RDP in China. The mutations of ATP1A3 have been previously reported in 51 patients including 6 large families and 16 other unrelated patients. A total of 14 different mutations in ATP1A3 gene with RDP have been reported to date, including 12 missense mutations, a 3-bp in-frame deletion, and a 3-bp in-frame insertion. The sporadic cases all had the typical clinical phenotypes of RDP, such as the abrupt onset of dysarthria, dysphagia, limb dystonia with bradykinesia, and postural instability. The symptoms of bulbar and arms were much more obvious. It was hard to diagnose RDP in a family because some patients had typical symptoms of RDP, while the others might experience from mild symptoms to no symptoms, which might be related to incomplete penetrance of RDP. Two cases carrying the same mutation as our patients also presented some overlapping phenotypes. Conclusion: The p. R756H heterozygous mutation in ATP1A3 gene is the pathogenic mutation of RDP, analysis of genotype-phenotype correlations of RDP will be very important and meaningful.

Reovirus as an oncolytic agent against experimental human malignant gliomas.

BACKGROUND: Reovirus is a naturally occurring oncolytic virus that usurps activated Ras-signaling pathways of tumor cells for its replication. Ras pathways are activated in most malignant gliomas via upstream signaling by receptor tyrosine kinases. The purpose of this study was to determine the effectiveness of reovirus as an experimental treatment for malignant gliomas. METHODS: We investigated whether reovirus would infect and lyse human glioma cell lines in vitro. We also tested the effect of injecting live reovirus in vivo on human gliomas grown subcutaneously or orthotopically (i.e., intracerebrally) in mice. Finally, reovirus was tested ex vivo against low-passage cell lines derived from human glioma specimens. All P values were two-sided. RESULTS: Reovirus killed 20 (83%) of 24 established malignant glioma cell lines tested. It caused a dramatic and often complete tumor regression in vivo in two subcutaneous (P =.0002 for both U251N and U87) and in two intracerebral (P =.0004 for U251N and P =.0009 for U87) human malignant glioma mouse models. As expected, serious toxic effects were found in these severely immunocompromised hosts. In a less immunocompromised mouse model, a single intratumoral inoculation of live reovirus led to a dramatic prolongation of survival (compared with control mice treated with dead virus; log-rank test, P<.0001 for both U251N and U87 cell lines). The animals treated with live virus also appeared to be healthier and gained body weight (P =.0001). We then tested the ability of reovirus to infect and kill primary cultures of brain tumors removed from patients and found that it killed nine (100%) of nine glioma specimens but none of the cultured meningiomas. CONCLUSIONS: Reovirus has potent activity against human malignant gliomas in vitro, in vivo, and ex vivo. Oncolysis with reovirus may be a potentially useful treatment for a broad range of human cancers.

[The role of PACAP protein in chronic sinusitis with or without nasal polyps].

Objective:To investigate the expression of PACAP protein in chronic rhinosinusitis without/with nasal polyps and refractory chronic rhinosinusitis.Method: Fifty-three patients with nasal polyps,70 cases with chronic sinusitis, 28 patients with refractory chronic rhinosinusitis and 20 control cases were enrolled for this study. The expression of PACAP protein was detected by immunochemistry.Result: ①PACAP protein were expressed in nasal epithelium，glandular epithelium and goblet cells；②The positive intensity of PACAP was" +", " +++", "－-+",and " ++" in nasal polyps, chronic rhinosinusitis, refractory chronic rhinosinusitis, and control group, respectively.Conclusion:PACAP protein mainly locates in nasal epithelium，glandular epithelium and goblet cells. Reduced expression of PACAP may be related with onset of chronic rhiniosinusitis without/with nasal polyps.

Downregulation of platelet-derived growth factor receptor-beta in Shp-2 mutant fibroblast cell lines.

The SH2-containing tyrosine phosphatase Shp-2 appears to function downstream of a variety of growth factor receptors and might play a positive role in cell proliferation. Here we report that expression of the beta subunit of platelet-derived growth factor receptor (PDGFR-beta) was specifically downregulated in mutant fibroblasts lacking a functional Shp-2, while the levels of PDGFR-alpha EGFR and IGFIR were not changed. PDGF-stimulated DNA synthesis and extracellular signal regulated kinase (Erk) activation was severely suppressed in mutant cells. RasGAP, that responds to activation of PDGFR-beta but not PDGFR-alpha, was not phosphorylated on tyrosine in mutant cells upon PDGF-treatment. Northern blot analysis failed to detect PDGFR-beta mRNA in mutant cells. The transcription initiation from the PDGFR-beta gene promoter was not significantly changed, but the half-life of its mRNA was shortened in Shp-2 mutant cells. These observations indicate that Shp-2 not only participates in transmission of signals from growth factor receptors but also plays a specific role in the control of the PDGFR-beta expression. We propose that this is an important mechanism for the positive control of cell proliferation by Shp-2.

Leptin restores euglycemia and normalizes glucose turnover in insulin-deficient diabetes in the rat.

Leptin has been shown to improve insulin sensitivity and glucose metabolism in normoinsulinemic healthy or obese rodents. It has not been determined whether leptin may act independently of insulin in regulating energy metabolism in vivo. The present study was designed to examine the effects of leptin treatment alone on glucose metabolism in insulin-deficient streptozotocin (STZ)-induced diabetic rats. Four groups of STZ-induced diabetic rats were studied: 1) rats treated with recombinant methionine murine leptin subcutaneous infusion with osmotic pumps for 12-14 days (LEP; 4 mg x kg(-1) x day(-1), n = 10); 2) control rats infused with vehicle (phosphate-buffered saline) for 12-14 days (VEH; n = 10); 3) pair-fed control rats given a daily food ration matching that of LEP rats for 12-14 days (PF; n = 8); and 4) rats treated with subcutaneous phloridzin for 4 days (PLZ; 0.4 g/kg twice daily, n = 10). Phloridzin treatment normalizes blood glucose without insulin and was used as a control for the effect of leptin in correcting hyperglycemia. All animals were then studied with a hyperinsulinemic-euglycemic clamp (6 mU x kg(-1) x min(-1). Our study demonstrates that leptin treatment in the insulin-deficient diabetic rats restored euglycemia, minimized body weight loss due to food restriction, substantially improved glucose metabolic rates during the postabsorptive state, and restored insulin sensitivities at the levels of the liver and the peripheral tissues during the glucose clamp. The effects on glucose turnover are largely independent of food restriction and changes in blood glucose concentration, as evidenced by the minimal improvement of insulin action and glucose turnover parameters in the PF and PLZ groups. Our results suggest that the antidiabetic effects of leptin are achieved through both an insulin-independent and an insulin-sensitizing mechanism.

Opposite effects of acute hypoglycemia and acute hyperglycemia on glucose transport and glucose transporters in perfused rat skeletal muscle.

This study was undertaken to characterize the effects of glycemia per se (glucose effectiveness) on muscle glucose transport. Isolated rat hindlimbs were perfused in situ for 2 h with perfusate containing either low (2 mmol/l, n = 7), normal (6.5 mmol/l, n = 6), or high (20 mmol/l, n = 6) concentrations of glucose, without insulin, to simulate hypo-, eu-, and hyperglycemic conditions. The effect of varying glucose concentrations on muscle glucose transport was assessed by an ensuing 30-min perfusion with 5.5 mmol/l glucose perfusate without insulin. The 2-h of low glucose perfusion induced significant increases in both muscle glucose clearance (approximately 2.3-fold, P < 0.01) and plasma membrane GLUT4 content (approximately 20%, P < 0.05) relative to normal. In contrast, high glucose perfusion decreased glucose clearance (approximately 1.7-fold, P < 0.01) and plasma membrane GLUT4 content (approximately 20%, P < 0.05). Glucose extraction during the following 30-min perfusion was 2.5-fold greater (P < 0.0001) in the low group and threefold less (P < 0.0001) in the high group, relative to normal. 2-[3H]deoxyglucose-6-phosphate content in both red (soleus) and white (extensor digitorum longus) muscles increased approximately twofold after 2 h of low glucose perfusion (P < 0.0001) and decreased > or =2-fold after high glucose perfusion (P < 0.0001), relative to normal. It is concluded that glycemia regulates glucose transport in skeletal muscle independently of insulin, achieved at least partially via changes in plasma membrane GLUT4. We propose that high glucose levels can acutely downregulate GLUT4 and glucose clearance, thus limiting excessive glucose uptake in muscle. Conversely, low glucose-induced upregulation of muscle glucose clearance and GLUT4 can compensate for reduced glucose availability in the circulation.

Importance of substrate changes in the decrease of hepatic glucose cycling during insulin infusion and declining glycemia in the depancreatized dog.

We wished to determine whether the elevated glucose cycling (GC) between glucose and glucose-6-phosphate (G<-->G6P) in diabetes can be reversed with acute insulin treatment. In six insulin-deprived, anesthetized, depancreatized dogs, insulin was infused for 6-9 h at a starting dose of 45-150 pmol.kg-1.min-1 to normalize plasma glucose from 23.9 +/- 1.4 to 5.0 +/- 0.4 mmol/l and gradually decreased to and maintained at a basal rate (1.7 +/- 1.0 pmol.kg-1.min-1) during the last 3 h. GC, measured with [2-3H]- and [6-3H]glucose, fell markedly from 15.3 +/- 2.7 and normalized at 1.3 +/- 0.6 mumol.kg-1.min-1 (P < 0.001). This occurred because total hepatic glucose output fell much more (from 41.2 +/- 3.1 to 11.6 +/- 1.2) than did glucose production (from 25.9 +/- 1.9 to 10.3 +/- 1.0 mumol.kg-1.min-1) (both P < 0.01). Freeze-clamped liver biopsies were taken at timed intervals for measurements of hepatic enzymes and substrates. The elevated hepatic hexose-6-phosphate levels decreased with insulin infusion (151 +/- 24 vs. 71 +/- 13 nmol/g, P < 0.01). Maximal activities of glucose-6-phosphatase (G6Pase) (from 17.6 +/- 0.8 to 19.6 +/- 2.6 U/g) and glucokinase (from 1.1 +/- 0.2 to 1.0 +/- 0.2 U/g) did not change. Insulin infusion resulted in a threefold increase (P < 0.05) in the activity of glycogen synthase (active form), but had no effect on hepatic glycogen content.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-independent acute restoration of euglycemia normalizes the impaired glucose clearance during exercise in diabetic dogs.

At rest and during exercise, chronic hyperglycemia, high free fatty acid (FFA) oxidation, and insulin deficiency in diabetes are well known to impair glucose clearance (metabolic clearance rate [MCR]). The effect of acute restoration of glycemia per se on MCR has been less well characterized. We therefore studied normal and alloxan-diabetic dogs both at rest and during exercise, as diabetic hyperglycemic or after acutely induced euglycemia (<160 min) generated by infusion of either insulin or phlorizin. Glucose uptake was similar under hyperglycemic and normoglycemic conditions both at rest and during exercise, indicating a precise balance between the mass effect of glucose and decreased MCR. Rest and exercise MCR was fourfold lower under conditions of hyperglycemia, but insulin-independent restoration of euglycemia improved basal MCR threefold and normalized MCR during exercise. High FFA turnover did not affect glucose uptake but was correlated with plasma lactate concentrations (r = 0.72, P < 0.001), suggesting that muscle fuel requirements are controlled by glucose oxidation and not uptake. We conclude that in alloxan-diabetic dogs, the impaired MCR may be an adaptive phenomenon because correction of hyperglycemia corrects MCR despite partial insulin deficiency and high FFA turnover. We speculate that constant glucose uptake despite hyperglycemia in diabetes may protect the muscle from excessive exposure to glucose.

The effect of pioglitazone on hepatic glucose uptake measured with indirect and direct methods in alloxan-induced diabetic dogs.

Pioglitazone, a thiazolidinedione derivative, ameliorates hyperglycemia by augmenting peripheral glucose disposal and suppressing hepatic glucose production in diabetic animals. However, the effect of this agent on hepatic glucose uptake has not been explored. To determine this, experiments were conducted in alloxan-induced diabetic dogs with (pioglitazone group, n = 7) or without (control group, n = 5) a 10-day oral treatment with pioglitazone (1 mg x kg(-1) x day(-1)). A euglycemic-hyperinsulinemic (insulin infusion rate 25.2 pmol x kg(-1) x min(-1)) clamp was maintained by adjusting the peripheral glucose infusion rate (GIR). After a 60-min basal period (period I), portal glucose infusion (Pinf, 33.3 micromol x kg(-1) x min(-1)) was administered for 120 min (period II). This was followed by a 60-min recovery period (period III). Arterial insulin levels were kept stable in the supraphysiological range throughout the experiment (1,623 +/- 52, pioglitazone group; 1,712 +/- 52 pmol/l, C group). There was no significant difference in whole-body glucose utilization determined by [3-3H]glucose between the pioglitazone and C groups in period I (68.4 +/- 2.8 vs. 70.1 +/- 2.8 micromol x kg(-1) x min(-1), respectively) and period III (81.2 +/- 5.0 vs. 74.5 +/- 3.3 micromol x kg(-1) x min(-1), respectively). Net hepatic glucose uptake (NHGU) determined by arteriovenous difference method was approximately zero in the basal period (-0.7 +/- 1.1, pioglitazone group; 0.1 +/- 1.2 micromol x kg(-1) x min(-1), C group). In period II, hepatic glucose uptake, determined by the changes in GIR, was significantly higher in the pioglitazone group (6.5 +/- 0.6 micromol x kg(-1) x min(-1)) than in the C group (-0.4 +/- 0.6 micromol x kg(-1) x min(-1), P < 0.001). This observation was also confirmed by NHGU during portal glucose infusion (6.9 +/- 1.4 vs. 2.1 +/- 1.8 micromol x kg(-1) x min(-1), pioglitazone vs. C, respectively; P < 0.025). We conclude that pioglitazone treatment enhances hepatic glucose uptake during portal glucose loading in alloxan-induced diabetic dogs. However, in hyperinsulinemic conditions, pioglitazone does not enhance the already high peripheral glucose uptake.

Beta-blockade, but not normoglycemia or hyperinsulinemia, markedly diminishes stress-induced hyperglycemia in diabetic dogs.

Stress-induced hyperglycemia can lead to significant deterioration in glycemic control in individuals with diabetes. Previously, we have shown in normal dogs that, after intracerebroventricular (ICV) administration of carbachol (a model of moderate stress), increases in both the metabolic clearance rate (MCR) of glucose and endogenous glucose production (GP) occur. However, in hyperglycemic diabetic dogs subjected to the same stress, the MCR of glucose does not increase and glycemia therefore markedly deteriorates because of stimulation of GP. Our aims were to determine the following: 1) whether insulin-induced acute normalization of glycemia, with or without beta-blockade, would correct glucose clearance and prevent the hyperglycemic effect of stress, and 2) whether hyperinsulinemia per se could correct these abnormalities. Stress was induced by ICV carbachol in 27 experiments in five alloxan-administered diabetic dogs subjected to the following protocols in random order: 1) basal insulin infusion (BI) to restore normoglycemia; 2) basal insulin infusion with beta-blockade (BI+block); 3) normoglycemic-hyperinsulinemic clamp with threefold elevation of insulin above basal (3x BI); and 4) normoglycemic-hyperinsulinemic clamp with fivefold elevation of insulin above basal (5 x BI). The BI+block protocol fully prevented stress-induced hyperglycemia, both by increasing MCR (deltaMCR at peak: 0.72 +/- 0.25 ml x kg(-1) x min(-1) vs. no change in BI, P < 0.05) and by diminishing the stress-induced increment in GP observed in BI (deltaGP at peak: 3.72 +/- 0.09 micromol x kg(-1) x min(-1) for BI+block vs. 14.10 +/- 0.31 micromol x kg(-1) x min(-1) for BI, P < 0.0001). In contrast, 3x BI and 5x BI treatments with normoglycemic-hyperinsulinemic clamps proportionately increased basal MCR at baseline, but paradoxically were not associated with an increase in MCR in response to stress, which induced a twofold increase in GP. Thus, in alloxan-administered diabetic dogs, stress increased GP but not MCR, despite normalization of glycemia with basal or high insulin. In contrast, beta-adrenergic blockade almost completely restored the metabolic response to stress to normal and prevented marked hyperglycemia, both by limiting the rise in GP and by increasing glucose MCR. We conclude that acute normalization of glycemia with basal insulin or hyperinsulinemia does not prevent hyperglycemic effects of stress unless accompanied by beta-blockade, and we speculate that short-term beta-blockade may be a useful treatment modality under some stress conditions in patients with diabetes.