Treatment of primary or recurrent non-resectable pancreatic cancer with proton beam irradiation combined with gemcitabine-based chemotherapy.

BACKGROUND: Pancreatic cancer accounts for around 4.6% of cancers deaths worldwide per year. Despite many advances in treatment regimes, the prognosis is still poor. Only 20% of tumors are primarily resectable. Recurrences-both with distant metastasis as well as locoregional-are frequent. For patients with primary nonresectable localized disease or localized recurrences, we offered chemoradiation to achieve local control over a long period of time. We here report our results on combined chemoradiation of pancreatic tumors and local recurrences using proton beam therapy. MATERIALS AND METHODS: We report on 25 patients with localized nonresectable pancreatic cancer (15 patients) or local recurrent disease (10 patients). All patients were treated with combined proton radiochemotherapy. Overall survival, progression-free survival, local control, and treatment-related toxicity were analyzed using statistically methods. RESULTS: Median RT dose was 54.0 Gy (RBE) for proton irradiation. The toxicity of treatment was acceptable. Four CTCAE grade III and IV adverse events (bone marrow disfunction, gastrointestinal [GI] disorders, stent dislocation, myocardial infarction) were recorded during or directly after the end of radiotherapy; two of them were related to combined chemoradiation (bone marrow disfunction, GI disorders). Six weeks after radiotherapy, one additional grade IV toxicity was reported (ileus, caused by peritoneal carcinomatosis, not treatment related). The median progression-free survival was 5.9 months and median overall survival was 11.0 months. The pretherapy CA19­9 level was a statistically significant prognostic factor for enhanced overall survival. Local control at 6 months and 12 months were determined to be 86% and 80%, respectively. CONCLUSION: Combined proton chemoradiation leads to high local control rates. Unfortunately, PFS and OS are driven by distant metastasis and were not improved compared to historical data and reports. With this in mind, enhanced chemotherapeutical regimes, in combination with local irradiation, should be evaluated.

Monocentric evaluation of Ki-67 labeling index in combination with a modified RPA score as a prognostic factor for survival in IDH-wildtype glioblastoma patients treated with radiochemotherapy.

PURPOSE: The prognosis for glioblastoma patients remains dismal despite intensive research on better treatment options. Molecular and immunohistochemical markers are increasingly being investigated as understanding of their role in disease progression grows. O(6)-methylguanine-DNA methyltransferase (MGMT) promoter methylation has been shown to have prognostic and therapeutic relevance for glioblastoma patients. Other markers implicated in tumor formation and/or malignancy are p53, Alpha thalassemia/mental retardation syndrome X-linked (ATRX), Epidermal Growth Factor Receptor splice variant III (EGFRvIII), and Ki-67, with loss of nuclear ATRX expression and lower Ki-67 index being associated with prolonged survival. For p53 and EGFRvIII the data are contradictory. Our aim was to investigate the markers mentioned above regarding progression-free (PFS) and overall survival (OS) to evaluate their viability as independent prognostic markers for our patient collective. METHODS: In this retrospective study, we collected data on patients undergoing radiotherapy due to isocitrate dehydrogenase (IDH) wildtype glioblastoma at a single university hospital between 2014 and 2020. RESULTS: Our findings confirm Ki-67 labeling index ≤ 20% as an independent prognostic factor for prolonged PFS as well as MGMT promoter methylation for both prolonged PFS and OS, in consideration of age and Eastern Cooperative Oncology Group (ECOG) status, chemotherapy treatment, and total radiation dose for PFS as well as additionally sex, resection status, and receipt of treatment for progression or recurrence for OS. Additionally, Ki-67 labeling index ≤ 20% showed a significant correlation with prolonged OS in univariate analysis. Modification of the recursive partitioning analysis (RPA) score to include Ki-67 labeling index resulted in a classification with the possible ability to distinguish long-term-survivors from patients with unfavorable prognosis. CONCLUSION: MGMT promoter methylation and Ki-67 labeling index were independent predictors of survival in our collective. We see further studies pooling patient collectives to reach larger patient numbers concerning Ki-67 labeling index as being warranted.

Advantage of proton-radiotherapy for pediatric patients and adolescents with Hodgkin's disease.

Radiotherapy is frequently used in the therapy of lymphoma. Since lymphoma, for example Hodgkin's disease, frequently affect rather young patients, the induction of secondary cancer or other long-term adverse effects after irradiation are important issues to deal with. Especially for mediastinal manifestations numerous organs and substructures at risk play a role. The heart, its coronary vessels and cardiac valves, the lungs, the thyroid and, for female patients, the breast tissue are only the most important organs at risk. In this study we investigated if proton-radiotherapy might reduce the dose delivered to the organs at risk and thus minimize the therapy-associated toxicity. METHODS: In this work we compared the dose delivered to the heart, its coronary vessels and valves, the lungs, the thyroid gland and the breast tissue by different volumetric photon plans and a proton plan, all calculated for a dose of 28.8 Gy (EURO-NET-PHL-C2). Target Volumes have been defined by F18-FDG PET-positive areas, following a modified involved node approach. Data from ten young female patients with mediastinal lymphoma have been evaluated. Three different modern volumetric IMRT (VMAT) photon plans have been benchmarked against each other and against proton-irradiation concepts. For plan-evaluation conformity- and homogeneity-indices have been calculated as suggested in ICRU 83. The target volume coverage as well as the dose to important organs at risk as the heart with its substructures, the lungs, the breast tissue, the thyroid and the spinal cord were calculated and compared. For statistical evaluation mean doses to organs at risk were evaluated by non- parametric Kruskal-Wallis calculations with pairwise comparisons. RESULTS: Proton-plans and three different volumetric photon-plans have been calculated. Proton irradiation results in significant lower doses delivered to organ at risk. The median doses and the mean doses could be decreased while PTV coverage is comparable. As well conformity as homogeneity are slightly better for proton plans. For several organs a risk reduction for secondary malignancies has been calculated using literature data as reference. According to the used data derived from literature especially the secondary breast cancer risk, the secondary lung cancer risk and the risk for ischemic cardiac insults can be reduced significantly by using protons for radiotherapy of mediastinal lymphomas. CONCLUSION: Irradiation with protons for mediastinal Hodgkin-lymphoma results in significant lower doses for almost all organs at risk and is suitable to reduce long term side effects for pediatric and adolescent patients.

Human mRNA response to exercise and temperature.

The purpose of this research was to determine the mRNA response to exercise in different environmental temperatures. 9 recreationally active males (27±1 years, 77.4±2.7 kg, 13.5±1.5% fat, 4.49±0.15 L · min (-1) VO2 max) completed 3 trials consisting of 1 h cycling exercise at 60% Wmax followed by a 3 h recovery in the cold (7°C), room temperature (20°C), and hot (33°C) environments. Muscle biopsies were obtained pre, post, and 3 h post exercise for the analysis of glycogen and mRNA. Expired gases were collected to calculate substrate use. PGC-1α increased to a greater degree in the cold trial than in the room temperature trial (p=0.036) and the hot trial (p=0.006). PGC1-α mRNA was also higher after the room temperature trial than the hot trial (p=0.050). UCP3 and MFN2 mRNA increased with exercise (p<0.05), but were unaffected by temperature. COX was unaffected by exercise or temperature. Muscle glycogen decreased with exercise (p<0.05), but was no different among trials. Whole body VO2 was lower during exercise in the cold than exercise in the heat. However, VO2 was higher during recovery in the cold trial than in the room temperature and hot trials (p<0.05). This study presents evidence of PGC-1α temperature sensitivity in human skeletal muscle.

Post-160-km race illness rates and decreases in granulocyte respiratory burst and salivary IgA output are not countered by quercetin ingestion.

This study measured the influence of the flavonoid quercetin on immune changes and incidence rates of upper respiratory tract infections in ultramarathoners competing in the 160-km Western States Endurance Run. Sixty-three runners were randomized to quercetin and placebo groups, and under double-blinded methods ingested 1000 mg/day quercetin for 3 wks before, during, and 2 wks after the race. Thirty-nine of the 63 subjects (n = 18 for quercetin, n = 21 for placebo) finished the race and provided blood and saliva samples the morning before the race and 15 - 30 min postrace. Upper respiratory tract infections were assessed during the week before and the 2-wk period after the race using an illness symptom checklist. Race times did not differ significantly between quercetin and placebo groups. Significant pre- to postrace decreases were measured for natural killer cells (43 %), granulocyte respiratory burst activity (55 %), and salivary IgA output (48 %), and increases for neutrophil (288 %) and monocyte (211 %) cell counts, with no significant group differences. Postrace illness rates did not differ between groups. In conclusion, quercetin supplementation for 3 wks before and 2 wks after the Western States Endurance Run had no effect on illness rates, perturbations in leukocyte subset counts, or decreases in granulocyte respiratory burst activity and salivary IgA.

Effect of NSAID on muscle injury and oxidative stress.

Indirect markers of muscle damage and delayed onset muscle soreness were examined and correlated to changes in oxidative stress, plasma antioxidant potential, and use or nonuse of non-steroidal anti-inflammatory drugs in 60 ultra-marathoners following the Western States Endurance Run. Blood was collected prior to and immediately following the race and analyzed for muscle damage by creatine phosphokinase and oxidative stress by F (2)-isoprostanes, protein carbonyls, and lipid hydroperoxides and antioxidant potential by the ferric reducing ability of plasma. Subjects recorded delayed onset muscle soreness during the week following the race. Lipid hydroperoxide concentrations were unchanged, but F (2)-isoprostanes, protein carbonyls, ferric reducing ability of plasma, creatine phosphokinase, and delayed onset muscle soreness increased significantly postrace. Protein carbonyls were significantly higher postrace in nonsteroidal anti-inflammatory drug users versus nonusers (p < 0.05). However, there was no difference between users and non-users for all other markers. Postrace creatine phosphokinase concentrations were not correlated with oxidative stress markers but were correlated with changes in delayed onset muscle soreness. Based upon these findings, caution should be used when consuming nonsteroidal anti-inflammatory drugs during ultra distance events.

Carbohydrate effect: hormone and oxidative changes.

Carbohydrate administration during exercise diminishes stress hormone release, but the relationship of these hormones with oxidative stress has not been examined. Fifteen subjects functioned as their own controls and ingested carbohydrate (6 %) or placebo in a randomized design while cycling for 2.5-h ( approximately 75 % V.O (2peak)). Blood and skeletal muscle samples were collected 30 min pre-exercise, immediately post-exercise, and 12-h post-exercise and analyzed for F (2)-isoprostanes, ferric reducing ability of plasma, glucose, insulin, cortisol, epinephrine, and muscle glycogen, respectively. Statistical design was a 2 (treatment) x 3 (time) repeated measures analysis of variance. Glucose, insulin, and ferric reducing ability of plasma were significantly higher and F (2)-isoprostanes, cortisol, and epinephrine significantly lower in carbohydrate versus placebo. The decrease in muscle glycogen was not different. During cycling exercise, oxidative stress appears to be heavily influenced by carbohydrate ingestion and increased stress hormones.

Immune changes: 2 h of continuous vs. intermittent cycling.

Immune changes following 2 h of intensive cycling with or without rest intervals were measured in trained cyclists (n = 12) who functioned as their own controls during two test sessions that were separated by two weeks. Subjects cycled for 2.0 h at approximately 64 % Watts(max) continuously (C) or with 3-min rest intervals (R) interspersed every 10 min (2.6 h total time), with the order of the sessions randomized. Blood samples were collected 30-min pre-exercise, and immediately and 1-h postexercise, and assayed for blood leukocyte subset counts, plasma IL-6, IL-10, IL-1ra, IL-8, PHA-induced lymphocyte proliferation, and natural killer cell activity (NKCA). Significant time effects were measured for all immune measures, but no significant differences in the pattern of change were found between C and R exercise trials. In conclusion, immune changes induced by 2 h of intense and prolonged exercise paralleled those measured when athletes rested 3 min every 10 min of exercise.

Relationship between salivary IgA secretion and upper respiratory tract infection following a 160-km race.

AIM: The relationship between salivary IgA secretion rate and upper respiratory tract infection (URTI) was studied in 155 ultramarathoners (126 males, 29 females, mean age 46.5+/-0.7 y) who had qualified to run the 160-km 2003 Western States Endurance Run. METHODS: Subjects provided saliva samples during registration, held the morning before the race, and within 5-10 minutes postrace (mean race time, 26.2+/-0.3 h). Unstimulated saliva was collected by expectoration for 4 minutes into 15-mL plastic, sterilized vials. Runners finishing the race and providing pre- and postrace saliva samples (n=106) turned in a health log specifying URTI episodes and severity of symptoms for the 2-week period following the race. RESULTS: The total volume of saliva that the runners was able to expectorate during sample collection decreased 51% postrace compared to prerace values (P<0.001). Saliva protein concentration increased 20% (P<0.001) while the saliva protein IgA concentration decreased 10% (P<0.05). Salivary IgA secretion rate decreased 46% when comparing pre- to postrace values (P<0.001). Twenty-four percent of the runners finishing the race and providing salivary samples reported an URTI episode lasting 2 days or longer during the 2-week period following the race (mean number of days with symptoms was 5.4+/-0.6 days). The decrease in salivary IgA secretion rate (pre- to postrace) was 53% greater in the 25 runners reporting URTI (-355+/-45 microg/min) compared to the 81 runners not reporting URTI (-232+/-37 microg/min), (P=0.04). CONCLUSIONS: In summary, nearly 1 in 4 runners reported an URTI episode during the 2-week period following a 160-km race, and the decrease in salivary IgA secretion rate was significantly greater in these runners compared to those not reporting URTI.

Influence of carbohydrate ingestion on immune changes after 2 h of intensive resistance training.

Thirty strength-trained subjects were randomized to carbohydrate (CHO) or placebo (Pla) groups and lifted weights for 2 h (10 exercises, 4 sets each, 10 repetitions, with 2- to 3-min rest intervals). Subjects received 10 ml x kg(-1) x h(-1) CHO (6%) or Pla beverages during the weight training bout. Blood, saliva, and vastus lateralis muscle biopsy samples were collected before and after exercise. Blood cell counts were determined, and plasma was analyzed for IL-6, IL-10, IL-1 receptor antagonist (IL-1ra), IL-8, and cortisol. Muscle was analyzed for glycogen content and relative gene expression of 13 cytokines (IL-1alpha, IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p35, IL-12p40, IL-15, IFN-gamma, TNF-alpha) by use of real-time quantitative RT-PCR. Significant but modest increases were measured for plasma IL-6, IL-10, IL-1ra, and IL-8, but the pattern of increase did not differ between CHO and Pla groups. The rate of decrease in muscle glycogen content did not differ between CHO and Pla (P = 0.463). Muscle cytokine mRNA was detected preexercise for IL-1beta, IL-6, IL-15, IL-8, and TNF-alpha, and of these, IL-1beta, IL-6, IL-8, and TNF-alpha were significantly increased after the 2-h weight training bout. The increase in mRNA (fold difference from preexercise) did not differ between CHO and Pla groups. In summary, CHO vs. Pla ingestion did not alter modest increases measured for plasma IL-6, IL-10, IL-1ra, and IL-8, and muscle gene expression for IL-1beta, IL-6, IL-8, and TNF-alpha in strength-trained subjects lifting weights intensively for 2 h.

Immune and oxidative changes during and following the Western States Endurance Run.

Changes in immune and oxidative stress parameters were measured in ultramarathon runners competing in the 160-km Western States Endurance Run. Forty-five runners agreed to provide blood and saliva samples the morning before the race event, at the 90-km aid station, and 5 - 10 min post-race. Upper respiratory tract infection (URTI) during the two-week period post-race was assessed retrospectively by telephone interviews. Forty subjects completed 90-km (race time, 13.1 +/- 0.3 h), and 31 completed the 160-km race event (27.0 +/- 0.4 h). The blood neutrophil and monocyte counts rose 249 % and 214 %, respectively, in the 31 finishers. Salivary IgA (sIgA) secretion rate decreased significantly from 508 +/- 40 micro g/min pre-race, to 287 +/- 39 micro g/min at 90-km, and 254 +/- 30 micro g/min post-race (50 % decrease). Significant increases were measured in cytokines at 90-km and post-race, with post-race IL-10 increasing 9.5-fold, IL-1ra 6.1-fold, IL-6 50.2-fold, and IL-8 2.5-fold over pre-race levels. Post-race indicators of oxidative stress, F (2)-isoprostane and lipid hydroperoxides, increased 33 % and 88 %, respectively. Pearson product-moment correlations revealed positive correlations at 90-km between F (2)-isoprostane and IL-6 (r = 0.31, p = 0.048), IL-10 (r = 0.31, p = 0.050), and IL-8 (r = 0.43, p = 0.005), but no other significant relationships between immune and oxidative stress indicators at 90-km and post-race. In the group of runners completing at least 90 km of the race, 26 % reported an URTI episode during the two-week period post-race. A low sIgA secretion rate at 90-km was the best predictor of post-race URTI (173 +/- 34 micro g/min in those who later acquired URTI compared to 325 +/- 40 micro g/min in those without URTI, p = 0.007). In conclusion, a modest correlation was found between cytokines and F (2)-isoprostane at 90-km when the greatest oxidative stress occurred, but no other significant correlations in immune and oxidative stress indicators during and following a 160-km ultramarathon race event were noted. About one in four ultramarathoners reported URTI during the two-week period post-race, and a low sIgA secretion rate mid-race best predicted URTI occurrence.

Carbohydrate ingestion influences skeletal muscle cytokine mRNA and plasma cytokine levels after a 3-h run.

Sixteen experienced marathoners ran on treadmills for 3 h at approximately 70% maximal oxygen consumption (Vo(2 max)) on two occasions while receiving 1 l/h carbohydrate (CHO) or placebo (Pla) beverages. Blood and vastus lateralis muscle biopsy samples were collected before and after exercise. Plasma was analyzed for IL-6, IL-10, IL-1 receptor agonist (IL-1ra), IL-8, cortisol, glucose, and insulin. Muscle was analyzed for glycogen content and relative gene expression of 13 cytokines by using real-time quantitative RT-PCR. Plasma glucose and insulin were higher, and cortisol, IL-6, IL-10, and IL-1ra, but not IL-8, were significantly lower postexercise in CHO vs. Pla. Change in muscle glycogen content did not differ between CHO and Pla (P = 0.246). Muscle cytokine mRNA content was detected preexercise for seven cytokines in this order (highest to lowest): IL-15, TNF-alpha, IL-8, IL-1beta, IL-12p35, IL-6, and IFN-gamma. After subjects ran for 3 h, gene expression above prerun levels was measured for five of these cytokines: IL-1beta, IL-6, and IL-8 (large increases), and IL-10 and TNF-alpha (small increases). The increase in mRNA (fold difference from preexercise) was attenuated in CHO (15.9-fold) compared with Pla (35.2-fold) for IL-6 (P = 0.071) and IL-8 (CHO, 7.8-fold; Pla, 23.3-fold; P = 0.063). CHO compared with Pla beverage ingestion attenuates the increase in plasma IL-6, IL-10, and IL-1ra and gene expression for IL-6 and IL-8 in athletes running 3 h at 70% Vo(2 max) despite no differences in muscle glycogen content.

Role of kallikrein-kininogen system in insulin-stimulated glucose transport after muscle contractions.

Serum proteins [molecular weight (MW) > 10,000] are essential for increased insulin-stimulated glucose transport after in vitro muscle contractions. We investigated the role of the kallikrein-kininogen system, including bradykinin, which is derived from kallikrein (MW > 10,000)-catalyzed degradation of serum protein kininogen (MW > 10,000), on this contraction effect. In vitro electrical stimulation of rat epitrochlearis muscles was performed in 1) rat serum +/- kallikrein inhibitors; 2) human plasma (normal or kallikrein-deficient); 3) rat serum +/- bradykinin receptor-2 inhibitors; or 4) serum-free buffer +/- bradykinin. 3-O-methylglucose transport (3-MGT) was measured 3.5 h later. Serum +/- kallikrein inhibitors tended (P = 0.08) to diminish postcontraction insulin-stimulated 3-MGT. Contractions in normal plasma enhanced insulin-stimulated 3-MGT vs. controls, but contractions in kallikrein-deficient plasma did not. Supplementing rat serum with bradykinin receptor antagonist HOE-140 during contraction did not alter insulin-stimulated 3-MGT. Muscles stimulated to contract in serum-free buffer plus bradykinin did not have enhanced insulin-stimulated 3-MGT. Bradykinin was insufficient for postcontraction-enhanced insulin sensitivity. However, results with kallikrein inhibitors and kallikrein-deficient plasma suggest kallikrein plays a role in this improved insulin action.

Absence of insulin receptor substrate-1 expression does not alter GLUT1 or GLUT4 abundance or contraction-stimulated glucose uptake by mouse skeletal muscle.

The purpose of this study was to determine the influence of insulin receptor substrate-1 (IRS-1) expression on GLUT1 and GLUT4 glucose transporter protein abundance, contraction-stimulated glucose uptake, and contraction-induced glycogen depletion by skeletal muscle. Mice (6 months old) from three genotypes were studied: wild-type (IRS-1(+/+)), heterozygous (IRS-1(+/-)) for the null allele, and IRS-1 knockouts (IRS-1(-/-)) lacking a functional IRS-1 gene. In situ muscle contraction was induced (electrical stimulation of sciatic nerve) in one hindlimb using contralateral muscles as controls. Soleus and extensor digitorum longus were dissected and 2-deoxyglucose uptake was measured in vitro. 2-Deoxyglucose uptake was higher in basal muscles (no contractions) from IRS-1(-/-) vs. both other genotypes. Contraction-stimulated 2-deoxyglucose uptake and glycogen depletion did not differ among genotypes. Muscle IRS-1 protein was undetectable for IRS-1(-/-) mice, and values were approximately 40 % lower in IRS-1(+/-) than in IRS-1(+/+) mice. No difference was found in IRS-1(+/+) compared to IRS-1(-/-) groups regarding muscle abundance of GLUT1 and GLUT4. Substantial reduction or elimination of IRS-1 did not alter the hallmark effects of contractions on muscle carbohydrate metabolism--activation of glucose uptake and glycogen depletion.

Genetic selection of mice for high voluntary wheel running: effect on skeletal muscle glucose uptake.

Effects of genetic selection for high wheel-running activity (17th generation) and access to running wheels on skeletal muscle glucose uptake were studied in mice with the following treatments for 8 wk: 1) access to unlocked wheels; 2) same as 1, but wheels locked 48 h before glucose uptake measurement; or 3) wheels always locked. Selected mice ran more than random-bred (nonselected) mice (8-wk mean +/- SE = 8,243 +/- 711 vs. 3,719 +/- 233 revolutions/day). Body weight was 5-13% lower for selected vs. nonselected groups. Fat pad/body weight was ~40% lower for selected vs. nonselected and unlocked vs. locked groups. Insulin-stimulated glucose uptake and fat pad/body weight were inversely correlated for isolated soleus (r = -0.333; P < 0.005) but not extensor digitorum longus (EDL) or epitrochlearis muscles. Insulin-stimulated glucose uptake was higher in EDL (P < 0.02) for selected vs. nonselected mice. Glucose uptake did not differ by wheel group, and amount of running did not correlate with glucose uptake for any muscle. Wheel running by mice did not enhance subsequent glucose uptake by isolated muscles.

Calorie restriction increases insulin-stimulated glucose transport in skeletal muscle from IRS-1 knockout mice.

Calorie restriction (CR), even for brief periods (4-20 days), results in increased whole-body insulin sensitivity, in large part due to enhanced insulin-stimulated glucose transport by skeletal muscle. Evidence suggests that the cellular alterations leading to this effect are postreceptor steps in insulin signaling. To determine whether insulin receptor substrate (IRS)-1 is essential for the insulin-sensitizing effect of CR, we measured in vitro 2-deoxyglucose (2DG) uptake in the presence and absence of insulin by skeletal muscle isolated from wild-type (WT) mice and transgenic mice lacking IRS-1 (knockout [KO]) after either ad libitum (AL) feeding or 20 days of CR (60% of ad libitum intake). Three muscles (soleus, extensor digitorum longus [EDL], and epitrochlearis) from male and female mice (4.5-6 months old) were studied. In each muscle, insulin-stimulated 2DG uptake was not different between genotypes. For EDL and epitrochlearis, insulin-stimulated 2DG uptake was greater in CR compared to AL groups, regardless of sex. Soleus insulin-stimulated 2DG uptake was greater in CR compared with AL in males but not females. The diet effect on 2DG uptake was not different for WT and KO animals. Genotype also did not alter the CR-induced decrease in plasma constituents (glucose, insulin, and leptin) or body composition (body weight, fat pad/body weight ratio). Consistent with previous studies in rats, IRS-1 protein expression in muscle was reduced in WT-CR compared with WT-AL mice, and muscle IRS-2 abundance was unchanged by diet. Skeletal muscle IRS-2 protein expression was significantly lower in WT compared with KO mice. These data demonstrate that IRS-1 is not essential for the CR-induced increase in insulin-stimulated glucose transport in skeletal muscle, and the absence of IRS-1 does not modify any of the characteristic adaptations of CR that were evaluated.

Interaction between BTBR and C57BL/6J genomes produces an insulin resistance syndrome in (BTBR x C57BL/6J) F1 mice.

Insulin resistance is a common syndrome that often precedes the development of noninsulin-dependent diabetes mellitus (NIDDM). Both diet and genetic factors are associated with insulin resistance. BTBR and C57BL/6J (B6) mice have normal insulin responsiveness and normal fasting plasma insulin levels. However, a cross between these two strains yielded male offspring with severe insulin resistance. Surprisingly, on a basal diet (6.5% fat), the insulin resistance was not associated with fasting hyperinsulinemia. However, a 15% fat diet produced significant hyperinsulinemia in the male mice (twofold at 10 weeks; P < .05). At 10 weeks of age, visceral fat contributed approximately 4.3% of the total body weight in the males versus 1.8% in females. In the males, levels of plasma triacylglycerol and total cholesterol increased 40% and 30%, respectively, compared to females. Plasma free fatty acid concentrations were unchanged. Oral glucose tolerance tests revealed significant levels of hyperglycemia and hyperinsulinemia 15 to 90 minutes after oral glucose administration in the male mice. This was particularly dramatic in males on a 15% fat diet. Glucose transport was examined in skeletal muscles in (BTBR x B6)F1 mice. In the nonhyperinsulinemic animals (females), insulin stimulated 2-deoxyglucose transport 3.5-fold in the soleus and 2.8-fold in the extensor digitorum longus muscles. By contrast, glucose transport was not stimulated in the hyperinsulinemic male mice. Hypoxia stimulates glucose transport through an insulin-independent mechanism. This is known to involve the translocation of GLUT4 from an intracellular pool to the plasma membrane. In the insulin-resistant male mice, hypoxia induced glucose transport as effectively as it did in the insulin-responsive mice. Thus, defective glucose transport in the (BTBR x B6)F1 mice is specific for insulin-stimulated glucose transport. This is similar to what has been observed in muscles taken from obese NIDDM patients. These animals represent an excellent genetic model for studying insulin resistance and investigating the transition from insulin resistance in the absence of hyperinsulinemia to insulin resistance with hyperinsulinemia.

An effector site that stimulates G-protein GTPase in photoreceptors.

Heterotrimeric G-proteins mediate between receptors and effectors, acting as molecular clocks. G-protein interactions with activated receptors catalyze the replacement of GDP bound to the alpha-subunit with GTP. alpha-Subunits then modulate the activity of downstream effectors until the bound GTP is hydrolyzed. In several signal transduction pathways, including the cGMP cascade of photoreceptor cells, the relatively slow GTPase activity of heterotrimeric G-proteins can be significantly accelerated when they are complexed with corresponding effectors. In the phototransduction cascade the GTPase activity of photoreceptor G-protein, transducin, is substantially accelerated in a complex with its effector, cGMP phosphodiesterase. Here we characterize the stimulation of transducin GTPase by a set of 23 mutant phosphodiesterase gamma-subunits (PDE gamma) containing single alanine substitutions within a stretch of the 25 C-terminal amino acid residues known to be primarily responsible for the GTPase regulation. The substitution of tryptophan at position 70 completely abolished the acceleration of GTP hydrolysis by transducin in a complex with this mutant. This mutation also resulted in a reduction of PDE gamma affinity for transducin, but did not affect PDE gamma interactions with the phosphodiesterase catalytic subunits. Single substitutions of 7 other hydrophobic amino acids resulted in a 50-70% reduction in the ability of PDE gamma to stimulate transducin GTPase, while substitutions of charged and polar amino acids had little or no effect. These observations suggest that the role of PDE gamma in activation of the transducin GTPase rate may be based on multiple hydrophobic interactions between these molecules.

Regulation of transducin GTPase activity in bovine rod outer segments.

The photoreceptor G-protein, transducin, belongs to the class of heterotrimeric GTP-binding proteins that transfer information from activated seven-span membrane receptors to effector enzymes or ion channels. Like other G-proteins, transducin acts as a molecular clock. It is activated by photoexcited rhodopsin which catalyzes the exchange of transducin-bound GDP for GTP and then stays active until bound GTP is hydrolyzed by an intrinsic GTPase activity. Our previous study on the components of the amphibian phototransduction cascade (Arshavsky, V. Y., and Bownds, M. D. (1992) Nature 357, 416-417) has shown that transducin GTPase can be significantly accelerated by the target enzyme, cGMP phosphodiesterase (PDE), and more specifically its gamma-subunit (PDE gamma). Here we report that an analogous mechanism is present in bovine photoreceptors. Addition of recombinant PDE gamma to the test photoreceptor membranes which retain transducin but are depleted of endogenous PDE causes a significant acceleration of transducin GTPase activity. A similar effect was observed with the PDE holoenzyme, but not with the complex of PDE alpha- and beta-subunits prepared by a limited proteolysis of PDE with trypsin. The activating effect of PDE gamma is increased as test membrane concentration increases, exceeding 20-fold at rhodopsin concentrations over 80 microM and approaching the rate of the photoresponse turnoff. This suggests either that photoreceptor membranes contain a further factor which is essential for PDE-dependent regulation of transducin-bound GTP hydrolysis or that components of the phototransduction cascade interact in a cooperative manner. We also report that the GTPase-activating epitope is located within the C-terminal third of PDE gamma: the peptide corresponding to the 25 C-terminal amino acid residues of PDE gamma can accelerate transducin GTPase almost as well as the full-length PDE gamma. A part of the GTPase activating epitope is located within the 3 C-terminal amino acid residues: the truncation PDE gamma mutant lacking these residues accelerates transducin GTPase considerably less than the whole length PDE gamma.

Rod outer segment structure influences the apparent kinetic parameters of cyclic GMP phosphodiesterase.

Cyclic GMP hydrolysis by the phosphodiesterase (PDE) of retinal rod outer segments (ROS) is a key amplification step in phototransduction. Definitive estimates of the turnover number, kcat, and of the Km are crucial to quantifying the amplification contributed by the PDE. Published estimates for these kinetic parameters vary widely; moreover, light-dependent changes in the Km of PDE have been reported. The experiments and analyses reported here account for most observed variations in apparent Km, and they lead to definitive estimates of the intrinsic kinetic parameters in amphibian rods. We first obtained a new and highly accurate estimate of the ratio of holo-PDE to rhodopsin in the amphibian ROS, 1:270. We then estimated the apparent kinetic parameters of light-activated PDE of suspensions of disrupted frog ROS whose structural integrity was systematically varied. In the most severely disrupted ROS preparation, we found Km = 95 microM and kcat = 4,400 cGMP.s-1. In suspensions of disc-stack fragments of greater integrity, the apparent Km increased to approximately 600 microM, though kcat remained unchanged. In contrast, the Km for cAMP was not shifted in the disc stack preparations. A theoretical analysis shows that the elevated apparent Km of suspensions of disc stacks can be explained as a consequence of diffusion with hydrolysis in the disc stack, which causes active PDEs nearer the center of the stack to be exposed to a lower concentration of cyclic GMP than PDEs at the disc stack rim. The analysis predicts our observation that the apparent Km for cGMP is elevated with no accompanying decrease in kcat. The analysis also predicts the lack of a Km shift for cAMP and the previously reported light dependence of the apparent Km for cGMP. We conclude that the intrinsic kinetic parameters of the PDE do not vary with light or structural integrity, and are those of the most severely disrupted disc stacks.