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Heat Shock Prevents Insulin Resistance–Induced Vascular Complications by Augmenting Angiotensin-(1-7) Signaling
We have investigated the role of heat shock (HS) in preventing insulin resistance–induced endothelial dysfunction. To the best of our knowledge, we report here for the first time that insulin resistance inhibits vascular HS protein (HSP) 72 expression. HS treatment (41°C for 20 min) restored the HSP72 expression. High-fat diet (HFD)–fed, insulin-resistant rats show attenuated angiotensin (ANG)-(1-7)–induced vasodilator effect, endothelial nitric oxide synthase (eNOS) phosphorylation, AMP-activated protein kinase phosphorylation, and sirtuin 1 (SIRT1) expression. Interestingly, HS prevented this attenuation. We also provide the first evidence that HFD-fed rats show increased vascular DNA methyltransferase 1 (DNMT1) expression and that HS prevented this increase. Our data show that in HFD-fed rats HS prevented loss in the expression of ANG-(1-7) receptor Mas and ACE2, which were responsible for vascular complications. Further, the inhibition of eNOS (l-NG-nitro-l-arginine methyl ester), Mas (A-779), and SIRT1 (nicotinamide) prevented the favorable effects of HS. This suggests that HS augmented ANG-(1-7) signaling via the Mas/eNOS/SIRT1 pathway. Our study, for the first time, suggests that induction of intracellular HSP72 alters DNMT1 expression, and may function as an epigenetic regulator of SIRT1 and eNOS expression. We propose that induction of HSP72 is a novel approach to prevent insulin resistance–induced vascular complications.
Insulin resistance is a prominent component of metabolic syndrome, which is characterized by obesity, hypertension, and atherosclerosis. A reciprocal relationship has been established between insulin resistance and endothelial dysfunction, which may lead to cardiovascular disorders (1). Apart from the metabolic actions, insulin performs various important hemodynamic functions such as peripheral vasodilation and increased regional blood flow via stimulation of nitric oxide (NO). Therefore, anything that impairs insulin action is expected to result in endothelial dysfunction and vice versa (2). Insulin has been shown to induce endothelial-dependent vasodilation without affecting endothelium-independent vasodilation. We and others (3,4) have reported that insulin resistance impairs endothelium-dependent vasodilation.
Several studies have pointed out active involvement of renin-angiotensin system (RAS) in cardiovascular disorders and insulin resistance (5,6). RAS further splits in two, as follows: angiotensin (ANG) II/ACE1 and ANG-(1-7)/ACE2 axis. ANG-(1-7), acting through receptor Mas (G-protein–coupled), counter-regulates the actions of ANG II. Mas receptor dimerizes with AT1 receptor and inhibits ANG II signaling, suggesting direct interaction of ANG II and the ANG-(1-7) axis (7). ANG-(1-7) promotes vasodilation and inhibits cell proliferation thrombosis, hence opposing the effects of ANG II (8,9). In endothelial cells, ANG-(1-7) inhibited ANG II–induced c-Src, extracellular signal–related kinase 1/2, and NADPH oxidase by activating SHP-2 phosphatase (10). ANG-(1-7) also inhibits the negative regulation of ANG II on insulin-induced AKT and NO production, suggesting the crucial role of ANG-(1-7) in maintaining insulin sensitivity (11). ANG-(1-7) and Mas agonist AVE0991 have shown beneficial effects in atherosclerosis and hypertension (12,13). Long-term treatment with ANG-(1-7) improved glucose uptake and insulin resistance in rats. ANG-(1-7) has also been found to be effective in mitigating diabetes complications like nephropathy and memory impairment (14,15). ANG-(1-7) is produced mostly from ANG II, and this reaction is catalyzed by ACE2. Overexpression of ACE2 or ACE1 inhibition with captopril is an effective way to increase ANG-(1-7) expression in tissues and circulation. ACE2 overexpression ameliorated the formation of atherosclerotic plaques, cardiac hypertrophy, and glomerulosclerosis, and this effect, in part, is mediated through ANG-(1-7) (16). Therefore, an imbalance between the ANG II/ACE1 axis and the ANG-(1-7)/ACE2 axis in the RAS has emerged as a common denominator in cardiovascular disorders (17).
It has been reported that endothelial NO synthase (eNOS) protein expression and NO release are decreased in clinically relevant human atherosclerosis (18). It has also been reported that loss in the expression of eNOS accelerated atheroma formation (19,20). Recently, Sansbury et al. (21) provided striking evidence that eNOS overexpression reduces adiposity in rodents fed with a high-fat diet (HFD). They have reported that eNOS activation in adipose and conduit or resistant vessels has profound clinical significance. As far as the stress response is concerned, calorie restriction or exercise training has also been shown to increase eNOS expression and NO bioavailability, which are implicated in mitochondrial biogenesis (22). Therefore, activated eNOS not only improves endothelial dysfunction but also alters processes such as adipocyte phenotype and mitochondrial biogenesis. Hence, eNOS appears to be much wiser than we thought. Hence, we hypothesized that in our model of insulin resistance eNOS expression might be altered.
Over the years, from the discovery of heat shock (HS) response, several studies pointed out the protective role of HS protein (HSP) 72 in various disorders like cardiac ischemia, Huntington disease, hypertension, and diabetes (23–25). In the study by Bruce et al. (26), correlation between decreased HSP72 expression and the degree of insulin resistance has been demonstrated. Further, diabetes has been shown to limit HSP72 expression in various tissues (25). These studies suggest that alteration in HSP expression is involved in the pathogenesis of insulin resistance. Induction of HSP72 by HS, transfection, or mild electrical stimulation prevented the insulin resistance in skeletal muscle and recently in monocytes (27–29). Several hypotheses for the beneficial effects of HSPs, such as inhibition of stress-activated kinases (Jun NH2-terminal kinase), reduction in oxidative stress, and nuclear factor-κβ (NF-κβ) activation have been proposed, but the mechanism is still poorly understood (29).
Epigenetics includes the stable alteration in DNA and histone proteins responsible for cell- and tissue-specific gene expression. DNA methylation is perhaps one of the most widely studied epigenetic phenomenon (30). DNA methylation, at CpG dinucleotides, has been implicated in a number of processes, such as transcriptional regulation, chromatin structure, and cancer pathogenesis. A growing body of evidence shows an inverse correlation between transcription and DNA methylation, and is marked as a gene-silencing signal (31). DNA methyltransferase (DNMT) 1 is an abundantly expressed DNMT that is essential for the maintenance of methylation patterns and the silencing of tumor suppressor genes (32). DNMT1 has been shown to be activated in proatherogenic conditions and to be responsible for the repression of genes like estrogen receptor alpha (ER-α) (33). But there are no reports depicting an association between HS and DNMT1 expression and its role in regulating the key molecules responsible for endothelial dysfunction in insulin resistance.
Chen et al. (23,34) for the first time reported the presence of cross talk between HSPs and RAS. Mild heat treatment (41°C) was able to decrease the ANG II–induced inflammation and hypertension. On the contrary, heat treatment at 45°C augmented ANG II–induced contraction and oxidative stress, suggesting the hormetic effect of HS (35). These studies determined the effect of HS on the vasoconstrictor arm, ANG II, of RAS. However, the vasoprotective arm of RAS, ANG-(1-7), has not been investigated. Therefore, we hypothesized that in vivo hormetic mild heat treatment may enhance the ANG-(1-7)/Mas/ACE2 axis and prevent endothelial dysfunction in insulin resistance.
Research Design and Methods
The 6- to 8-week-old male Sprague-Dawley rats (160–180 g) were procured from the Central Animal Facility of the National Institute of Pharmaceutical Education and Research. They were maintained under standard environmental conditions (temperature 20 ± 10°C; humidity 50 ± 10%; and 12 h light/dark cycle) with food and water available ad libitum. Animals were fed for 12 weeks with a normal pellet diet or an HFD (58 kcal% fat) (4). Experiments were conducted 48 h after the last heat or sham treatment, and rats were fasted 12 h before undergoing experimental procedures. All protocols were approved by the Institutional Animal Ethics Committee of the National Institute of Pharmaceutical Education and Research (IAEC13/14).
Drugs and Chemicals
ANG-(1-7), nicotinamide (NAM), PD123319, and l-NG-nitro-l-arginine methyl ester (L-NAME) were procured from Sigma-Aldrich. A-779 was procured from Bachem. Antibodies against HSP72 (1:3,000), phosphorylated (p) AMP-activated protein kinase (AMPK) (1:3,000), ACE2 (1:1,000), eNOS (1:2,000), p-eNOS (1:2,000), DNMT1 (1:1,000), and tubulin (1:3,000) were obtained from Santa Cruz Biotechnology; and HSP27 (1:3,000) and sirtuin 1 (SIRT1) (1:1,000) were obtained from Upstate Chemicals. Losartan was a generous gift from Novartis India. Unless specified, all other reagents were from Sigma-Aldrich.
In Vivo Heat Treatment
Animals were anesthetized with an intraperitoneal injection of ketamine/xylazine. Animals were wrapped in a homeothermic blanket (Harvard Apparatus). The blanket temperature was set at 45°C and the core body temperature was monitored using a rectal probe. The animal core temperature was maintained at 41°C for 20 min by opening and closing the blanket, as described before by Gupte et al. (29). For the sham treatment, rats were anesthetized, and their core temperature was maintained at 37°C. After the heat or sham treatment, the rats were administered 5 ml 0.9% saline i.p. to prevent dehydration.
Vascular Reactivity to ANG-(1-7) (ex vivo)
Aortic ring preparation and isometric tension measurement studies were performed with Panlab (Harvard Apparatus) and PowerLab data acquisition system (AD Instruments), as described previously (4). In brief, ANG-(1-7)–induced relaxation was studied by precontracting aortic rings with 100 nmol/L phenylephrine (PE) or 40 mmol/L KCl, after which the concentration-response curve (CRC) to ANG-(1-7) (1 nmol/L to 1 μmol/L) was recorded. To study the mechanism of ANG-(1-7)–induced relaxation, the second CRC to ANG-(1-7) was constructed. A total of two CRCs to ANG-(1-7) were constructed from each preparation with an interval of 2 h and with washing once every 15 min. The tissue was incubated with the vehicle, L-NAME (100 µmol/L), A-779 (10 µmol/L), PD123319 (10 µmol/L), and losartan (10 µmol/L) for 30 min, after which a second cumulative relaxation-response curve to ANG-(1-7) was elicited. Preliminary studies were performed to ascertain that the first and second cumulative relaxation-response curves were identical in the absence of any modulators.
In Vivo ANG-(1-7), NAM Treatment, and Blood Pressure Measurement
In the 12th week, animals were anesthetized and submitted to surgical procedure as soon as anesthesia was assured by loss of pedal and corneal reflexes. The jugular vein was cannulated with a PE catheter (AD Instruments), and in vivo stimulation of the aorta was performed by the injection of 200 μL of either saline or ANG-(1-7) (1,000 pmol/kg) into rats from each group; after 10 min, the aorta was isolated and frozen immediately for Western blotting. For selective antagonism of Mas receptor, A-779 (80 pmol/kg) was coadministered with ANG-(1-7) intravenously. In another independent study, NAM (200 mg/kg i.p.) was administered to HFD-fed rats before heat treatment to discover the role of SIRT1 (36). NAM was administered 2 h prior to heat treatment. Intra-arterial blood pressure was measured by a fluid-filled pressure transducer connected to a data acquisition system (AD Instruments) in controls, HFD-fed rats, and HFD-fed rats subjected to a single bout of HS.
Intraperitoneal Glucose Tolerance Test
After the last heat treatment, animals were anesthetized and given a glucose load of 2 g/kg i.p., and blood sampling was performed. The blood samples were taken at 0 (just before glucose administration), and 15, 30, 60, and 120 min after administration of the glucose load from all animals for estimation of glucose levels.
Western Blotting
Protein isolation and Western blotting were performed as previously described (4). Briefly, frozen aortic tissues were homogenized in liquid nitrogen and transferred to lysis buffer. Protein samples were resolved using 7.5%, 10%, and 14% SDS-PAGE, depending on the molecular weight of the desired proteins. These were then transferred to polyvinylidene fluoride membranes (PALL) and were analyzed with primary antibodies. The antibodies used were as follows: HSP72, p-AMPK, ACE2, eNOS, p-eNOS, HSP27, and SIRT1. Tubulin was used as the loading control. The antigen-primary antibody complexes were incubated with horseradish peroxidase–conjugated secondary antibodies and visualized using an enhanced chemiluminescence system and Hyperfilm ECL (Amersham). Blots were scanned and analyzed using ImageJ software.
Real-Time PCR
RNA was isolated from aorta using RNA extraction kit (Ambion). After reverse transcription with Superscript II (Invitrogen), real-time RT-PCR was performed on a Light Cycler 2 (Roche) using SYBR Master mix (Invitrogen) and the specific forward and reverse primers (Eurofins MWG Operon) (MAS: forward-ACGTCCCCAGACCAGTCA; reverse-TGAGGAGTTCTTGTGCTG). After amplification, a melting-curve analysis was performed to verify the specificity of the reaction. The 18S gene was used as an internal control, and results were determined by 2-ΔΔCt expressed as the fold change over control rats.
Histopathology and Immunohistochemistry
Rats were anesthetized and aorta, liver, muscle, white adipose tissue (WAT), and brown adipose tissue (BAT) were removed and stored in 10% formal saline. Paraffin blocks were prepared after completing the routine processing. Sections (3–5 mm) were prepared from paraffin blocks and stained with hematoxylin-eosin. Sections of liver, muscle, WAT, and BAT were deparaffinized with xylene, followed by antigen retrieval by heating in citrate buffer (10 mmol/L). The following rabbit polyclonal primary antibodies were used: anti-HSP72 (1:125). Polyvalent biotinylated goat anti-rabbit secondary antibody and a streptavidin-horseradish peroxidase system (Vectastain) were used to amplify the signals followed by detection with diaminobenzidine as a chromogen. Slides were counterstained with hematoxylin, dehydrated with alcohols and xylene, and mounted in DPX (Sigma-Aldrich). Histological images were captured by a charged-coupled device camera attached to a microscope (Model BX 51; Olympus, Tokyo, Japan). The intensity of the spot was graded from 1 to 4 (1, slight or no color; 2, very low color; 3, moderate brown color; and 4, very intense brown color). The immunohistochemistry score was expressed as the mean ± SEM for each experimental group.
Statistical Analysis
Results were expressed as the mean ± SEM, and n refers to the number of samples studied. Cumulative CRCs were analyzed by nonlinear curve fitting, and statistical differences between the means (maximal relaxation) were determined by one-way ANOVA using Prism software (version 5.0; GraphPad, San Diego, CA) for Windows. P < 0.05 was considered to be statistically significant.
Insulin Resistance: A State of Vascular HSP72 Deficiency
To evaluate whether HSP72 deficiency is involved in endothelial dysfunction associated with insulin resistance, we determined the expression profile of HSP72 in arteries of control and HFD-fed rats. We show that HSP72 is predominantly expressed in the aorta of healthy control rats. Aortic segments from HFD-fed, insulin-resistant rats showed markedly reduced expression of HSP72, as confirmed by immunohistochemical analysis (Fig. 1A and B) (P < 0.01). Western blotting analysis revealed that the expression of HSP72 was higher in arteries of control rats than in those of HFD-fed rats (Fig. 1C and D) (P < 0.05). These observations suggest that HSP72 may have a beneficial role in insulin resistance and its complications.
HS Treatment Improves Insulin Sensitivity and HSP72 Expression
Insulin resistance was developed in rats by feeding them an HFD for 12 weeks. From the first week of feeding, a subgroup of control and HFD-fed rats received mild heat treatment (i.e., HS; at 41°C once a week for 20 min), which is an established method for the induction of HSP72 (29). After the last HS treatment, animals were fasted overnight for the measurement of fasting glucose and insulin levels, and to perform an intraperitoneal glucose tolerance test (IPGTT). As expected, an HFD resulted in an elevation in body weight (Fig. 2A), fasting glucose (Fig. 2B) and insulin (Fig. 2C) levels, and insulin resistance, as analyzed by homeostatic model assessment for insulin resistance (HOMA-IR), compared with control rats (Fig. 2D). On the other hand, HFD-fed rats subjected to HS were protected from hyperglycemia, hyperinsulinemia, and elevated HOMA-IR. However, control rats subjected to HS showed no significant changes in fasting glucose and insulin levels, and HOMA-IR compared with control rats. Further, to check whether rats were protected against glucose load, we performed an IPGTT. The HFD-fed rats showed impaired glucose disposal, as observed by an increased area under the curve (AUC) than control rats (P < 0.01). However, HFD-fed rats subjected to HS showed improved glucose disposal and reduced AUC than HFD-fed rats. There was no change in the AUC in control rats and control rats subjected to heat treatment (Fig. 2E and F) (P < 0.05).
Previous studies have shown that mRNA and protein level of HSP72 are reduced in the skeletal muscle of patients with type 2 diabetes and insulin resistance (26,29). However, there are no reports regarding the inhibition of vascular HSP72 expression in insulin resistance. Here, we report that HSP72 expression is reduced significantly in the aortas of HFD-fed rats than in those of control rats (Fig. 2G–I) (P < 0.05). HS markedly restored the expression of HSP72 in HFD-fed rats (P < 0.05). We also determined the expression of HSP27. An HFD also decreased HSP27 expression to a certain extent, and HS slightly elevated it. In order to determine a correlation between HSP72 protein expression and fasting insulin levels, we performed regression analysis. Interestingly, we observed an inverse correlation between fasting insulin level and aortic HSP72 expression (Fig. 2J) (r2 = 0.65, P < 0.01). This observation was matched with earlier reports (26,37). Further, HFD-fed rats subjected to heat treatment showed decreased plasma cholesterol and triglyceride levels than rats fed an HFD alone (Supplementary Fig. 1A and B). HFD feeding also promoted adiposity because the weights of the visceral, mesentery, epididymal, and retroperitoneal fat pads were increased. HFD-fed rats subjected to HS were protected from any increase in adiposity index (Supplementary Fig. 1C–G). Also, HS redirected the fat from nonadipose tissue to adipose tissue. The liver weight and liver mass index were significantly increased by HFD feeding, and HS suppressed these changes (Supplementary Fig. 1H–M). Histopathological analysis revealed excessive fat deposition in hepatocytes and skeletal muscles by HFD feeding, and its reversal by heat treatment (Supplementary Fig. 2A). HFD feeding increased the adipocyte size and cholesterol deposition in WAT and BAT. HS significantly reduced the adipocyte size of WAT and BAT in HFD-fed rats (Supplementary Fig. 2B).
HS Treatment Improves ANG-(1-7)–Induced Vasodilator Responses
It is generally accepted that the tonic release of NO from the endothelium exerts vasculoprotective and cardioprotective effects in response to exercise or calorie restriction (38,39). We and others (1,4) have reported that the activation of ANG II is responsible for endothelial dysfunction in insulin resistance. Earlier, HS has been shown to suppress ANG II–induced hypertension and inflammation, but there are no reports suggesting a beneficial role of HS on the ANG-(1-7) pathway of RAS (23). Therefore, we determined vasodilator responses to ANG-(1-7) in insulin resistance condition. ANG-(1-7) induced concentration-dependent vasodilation in thoracic aorta. ANG-(1-7)–induced 40 ± 6% of maximum relaxation in control aortic rings. HFD feeding reduced the ANG-(1-7)–induced maximal relaxation to 22 ± 5% (Fig. 3A) (P < 0.01). This indicates that the protective action of ANG-(1-7) is masked by HFD-induced insulin resistance. Contrary to that, ANG-(1-7)–induced vasodilation was augmented to 39 ± 3% in aortic rings isolated from rats subjected to HS than HFD-fed rats (Fig. 3A) (P < 0.01). This indicates that heat treatment protected HFD-fed rats against reduced ANG-(1-7)–induced vasodilatory effect. The vasodilatory responses to ANG-(1-7) were comparable among control rats and control rats subjected to HS rat aortic rings (Fig. 3A). We also compared the sensitivity (negative logarithm value [pD2]) to ANG-(1-7) in control and HFD-fed rats subjected to either sham or heat treatment. The sensitivity was similar for all groups (Fig. 3B).
Further, we investigated the direct effect of HS on NO bioavailability and endothelial-dependent vasodilatation in insulin resistance. HS enhanced the vasodilation in response to carbachol (CCh) (Fig. 3C) (P < 0.05 vs. HFD), which was suppressed under HFD feeding (P < 0.001 vs. control). Vasodilation in response to CCh was endothelium-dependent, and therefore we assessed endothelium-independent vasodilation by using NO donor sodium nitroprusside (SNP). The relaxation responses to SNP were unaltered by either an HFD or HS and were comparable with control rings (Fig. 3D). This indicates an active involvement of endothelium in mediating the effects of HS. In order to determine NO bioavailability, we performed a Griess assay and a functional study with PE in the presence of L-NAME. HFD feeding resulted in decreased NO levels in the aorta compared with control rats, which corroborates our previous report (4). HS significantly elevated the NO levels obtained from the Griess method (Fig. 3E) (P < 0.05). We observed that the vascular reactivity to PE was increased in aortic rings obtained from rats subjected to heat treatment when preincubated with eNOS inhibitor L-NAME. This indicates that NO levels were elevated in aortas of rats subjected to HS (Fig. 3F) (P < 0.05). In the overall samples, the AUC for ANG-(1-7)–induced vasodilation was positively correlated to aortic HSP72 expression (Fig. 3G) (r2 = 0.71, P < 0.01). Gross histopathology of the aorta was performed by staining with hematoxylin-eosin. We did not observe any appreciable changes in microscopic characters of the aorta among all the groups (Supplementary Fig. 3). We have also checked the expression of inflammatory molecules such as p-NF-κβ and inhibitor of κB in the aorta. Interestingly we found that HFD increases p-NF-κβ and decreases inhibitor of κB expression. HS treatment was able to attenuate this effect and hence HFD-induced inflammation (Supplementary Fig. 4C and D). These observations matched with those of previous studies (23,29).
HS Treatment Improves In Vivo ANG-(1-7) Signaling
The study by Sampaio et al. (40) shows that ANG-(1-7) binds to Mas receptor, activates eNOS (S1177) phosphorylation, and releases NO. The AKT/protein kinase B pathway is also shown to be involved in eNOS activation and NO release (40). We reported, earlier in RESULTS, on ex vivo ANG-(1-7) signaling in isolated aortic rings, and we also assessed the effect of HFD and HS on the in vivo ANG-(1-7) signaling.
First, we checked ANG-(1-7)–induced S1177 phosphorylation of eNOS as it has been correlated with NO release. ANG-(1-7) significantly increased the p-eNOS expression within 10 min in control rats (P < 0.05). However, HFD feeding prevented the ANG-(1-7)–induced p-eNOS expression (P < 0.01).The p-eNOS expression was fully rescued by HS treatment (Fig. 4A, panels a and b). Further, we have assessed both the basal and ANG-(1-7)–stimulated AKT activation. The basal expression of p-AKT S473 was comparable in control and HFD-fed rats but increased significantly in HS rats (P < 0.05). ANG-(1-7) induced p-AKT expression in control rats (P < 0.001), whereas this effect was inhibited in HFD-fed rats. Interestingly, HS treatment enhanced the ANG-(1-7)–induced p-AKT expression significantly (P < 0.01) (Fig. 4A, panels c and d). Next, we determined the effect of ANG-(1-7) on p-AMPK expression. We found that ANG-(1-7) acute stimulation increased p-AMPK expression and that HFD feeding ablated this effect. HS restored the ANG-(1-7)–induced AMPK phosphorylation (Fig. 4A, panels e and f) (P < 0.01). Further, we analyzed HSP72 and SIRT1 expression after the acute stimulation of ANG-(1-7). It was found that neither HSP72 nor SIRT1 expression was altered after short stimulation of ANG-(1-7) (Fig. 4A, panels g and h). This indicates that acute stimulation of ANG-(1-7) was not able to increase the expression of SIRT1 or HSP72. Next, we checked the change in blood pressure by one-time HS exposure for 20 min in 3-month-old HFD-fed rats. Three months of HFD feeding significantly increased the systolic blood pressure compared with control rats on a normal pellet diet (P < 0.01). Heat treatment of HFD-fed rats showed reduced systolic blood pressure (P < 0.01 vs. HFD). The heart rate was comparable among the groups (Fig. 4H and I).
HS Treatment Upregulates ANG-(1-7) Receptor Mas and ACE2
ANG-(1-7) is produced mostly from ANG II by the enzyme ACE2, and its overexpression has been shown to increase the circulating and local levels of ANG-(1-7) (41). Accumulating evidence indicates that beneficial actions of ANG-(1-7) are mediated through the Mas receptor activation (40,41). Checking ANG-(1-7) receptor Mas expression by real-time PCR, we found that HFD feeding decreased the mRNA levels of Mas receptor gene (Fig. 5A) (P < 0.01). The rats subjected to HS treatment had higher expression of Mas receptor than HFD-fed rats (P < 0.01). We also determined the protein expression of ACE2 in all the groups. HFD feeding promoted a decrease in ACE2 expression, and HS treatment increased it (Fig. 5B and C) (P < 0.01).
HS Regulates DNMT1 Expression in HFD-Fed Rats
There exists an inverse correlation between transcription and DNA methylation, which is marked as a gene-silencing signal. The expression of eNOS, SIRT1, and HSP72 was reduced in insulin resistance, which indicates that activation of silencing marks enzymes such as DNMT1. Therefore, we assessed the expression of the enzyme responsible for altering DNA methylation, DNMT1, in the insulin resistance state and whether HS has any effect on it.
We found that DNMT1 expression was markedly increased in the aortas of HFD-fed rats compared with those of controls (P < 0.01) (Fig. 5D and E). Interestingly, heat treatment ameliorated this increase in the expression of DNMT1 in HFD-fed rats to a significant extent (P < 0.01). This interesting observation suggests that insulin resistance is a state of enriched DNA methylation, which might be playing a role in the regulation of many vasoprotective molecules like eNOS, SIRT1, and HSP72.
Role of Mas Receptors in Potentiating Vasodilator Effect Produced by ANG-(1-7) in HS Rats
We further sought to understand the mechanism of ANG-(1-7)–induced vasodilation in HFD-fed rats subjected to HS. To trace out the type of receptor involved, we incubated aortic rings with series of angiotensin receptor blockers for 30 min prior to visualizing ANG-(1-7) responses. Losartan (AT1 receptor blocker) and PD123319 (AT2 receptor blocker) did not affect ANG-(1-7)–induced relaxation responses in aortic rings isolated from rats subjected to HS. But A-779, a Mas receptor blocker, completely abolished the ANG-(1-7)–induced vasodilation (Fig. 6A) (P < 0.001). Further, we assessed ANG-(1-7)–induced eNOS and AKT phosphorylation in vivo, in the presence of A-779. ANG-(1-7) (1,000 pmol/kg)–induced eNOS (P < 0.01) and AKT phosphorylation (S473) (P < 0.01) were significantly inhibited by the administration of A-779 (80 pmol/kg) (Fig. 6B–D). This provides us with a plausible mechanism for ANG-(1-7)–induced vasodilation via Mas receptor.
Aortic rings obtained from HFD-fed rats subjected to HS were incubated either with or without L-NAME, an eNOS inhibitor. We observed that L-NAME promoted a decrease in ANG-(1-7)–induced relaxation responses in HFD+HS rats (Fig. 6E) (P < 0.01). ANG-(1-7) elicits some relaxation in the presence of L-NAME, which indicates that other factors, such as prostanoids or endothelium-dependent hyperpolarizing factor, are also involved in mediating the responses to ANG-(1-7) in the rat aorta.
Inhibition of SIRT1 Attenuates the Favorable Effects of Heat Treatment
Recent findings pointed out the role of SIRT1 in mediating HS response. SIRT1 deacetylates HS factor-1 (HSF-1) and promotes its occupancy of the HSP72 gene (36). The expression of SIRT1 was increased in rats subjected to HS. Hence, we became more interested in examining its role in mediating the effects of HS. We used NAM, which is an endogenous inhibitor of SIRT1 and acts through a salvage pathway (36).
NAM treatment indeed suppresses the induction of HSP72 in HFD-fed rats subjected to HS (Fig. 7A). It also prevented the increase in p-eNOS (S1177) level in HFD+HS rats (P < 0.01), which was very much comparable with the level in HFD-fed rats. Furthermore, the phosphorylation levels of AMPK and SIRT1 expression were also checked. As expected, HFD feeding reduced p-AMPK and SIRT1 expression compared with that in control rats, whereas HS ablated this effect. Treatment with NAM significantly suppressed the elevated p-AMPK expression in HFD+HS rats (P < 0.01). However, the expression of SIRT1 was not affected by NAM treatment in HFD+HS rats. This can be explained if we assume that NAM only inhibited the catalytic activity of SIRT1. Overall, all of these results show that p-AMPK, SIRT1, and p-eNOS are negatively correlated with insulin resistance (Fig. 7A–E).
At the functional level, NAM promoted a decrease in ANG-(1-7)–induced vasodilation in HS rats (Fig. 7F) (HFD+HS+NAM −21.6 ± 2.2; HFD+HS −35.1 ± 1.7; P < 0.05). This shows the direct involvement of SIRT1 in augmenting ANG-(1-7)–induced vasodilation. Likewise, NAM also suppressed the augmented CCh-induced vasodilation in aortic rings of HS rats (Fig. 7G) (HFD+HS+NAM −31.2 ± 2.2; HFD+HS −54.7 ± 2.4). Overall, NAM prevents the beneficial effects of HSP72 induction in HFD-fed rats, which highlights the presence of cross talk between SIRT1 and HSP72.
The data presented in this study provide compelling evidence that HSP72 (heat treatment) can be a potential target for treating endothelial dysfunction. We, for the first time, show that insulin resistance is a state of vascular HSP72 deficiency. HSP72, an inducible form of HSP, functions at the cellular level to protect cells against many stress conditions, such as temperature, hypoxia, and ischemia, and stabilizes the normal functions of proteins (42). In our study we show that vascular HSP72 expression is reduced in insulin resistance and is inversely correlated with circulating insulin levels. Our data support earlier findings demonstrating that there exists an inverse relationship between mRNA levels of HSP72 and insulin sensitivity in skeletal muscles of type 2 diabetic and obese patients (26,28,29). Previous studies have failed to address the mechanism of loss of HSP72 expression in insulin resistance (26,28). HSF-1 regulates the transcription of a large number of genes regulating protein homeostasis, including HSP72. Transient activation of HSF-1 by stress leads to conversion of the inactive monomer to DNA binding trimer (42). Westerheide et al. (36) have reported that SIRT1 deacetylates HSF-1 and enhances its affinity to DNA. This suggests that SIRTs are essential to induce HS response. In our study, we report that SIRT1 expression is reduced in insulin resistance, which correlates well with our observation of the reduced expression of HSP72 in insulin resistance. Apart from SIRT1, our data also highlight the role of AMPK in regulating HSF-1/HSP72 activity, either directly or indirectly.
Genome-wide promoter analysis of DNA methylation in patients with type 2 diabetes has suggested that hypermethylation of mitochondrial genes such as peroxisome proliferator–activated receptor γ coactivator-1-α promotes its silencing (43). Recently, a decrease in the expression of ER-α has been positively correlated with enhanced DNMT1 expression in the aorta of HFD-fed rats (29). Here we also report that DNMT1 expression is enhanced in the aortas of HFD-fed rats, and this increase was suppressed by HS. This interesting observation can explain the decreased expression of HSP72, SIRT1, and eNOS in insulin resistance, and its epigenetic regulation. However, additional studies are required to confirm that these genes are regulated by DNA methylation.
How Does HSP72 Induction Improve ANG-(1-7) Signaling?
HS has been shown to inhibit ANG II–induced hypertension and inflammation in the heart and aorta (23,34). This suggests the presence of cross talk between HS and RAS. We now report for the first time that insulin resistance impairs the ANG-(1-7) vasodilator effect, and the induction of HSP72 attenuates this impairment. Previously, we have reported that HFD upregulates ANG II/AT1 signaling, which leads to enhanced vasoconstriction in the aorta (4). It has been reported that the initial response in the ANG II/AT1 axis downregulates the ANG-(1-7)/Mas axis (44,45). We now report that HFD impairs ANG-(1-7)/Mas signaling the in aorta, and HS prevents this loss. This suggests that enhanced ANG II/AT1 signaling in the aortas of HFD-fed rats might be responsible for the downregulation of ANG-(1-7)/MAS/ACE2 axis and NO bioavailability. Overexpression of ACE2 or chronic ANG-(1-7) infusion has been shown to reduce blood pressure, and this effect may be due to the degrading of local ANG II and the improving of endothelial dysfunction (46,47). Previously, HS has been shown to suppress ANG II–induced hypertension in aorta (23). Together, the plausible explanation for the antihypertensive effect of HS may be the activation of ANG-(1-7) and the suppression of ANG II signaling in HFD-fed rats. The mechanism for the increase in vasodilator responses to ANG-(1-7) is probably due to enhanced downstream signaling of ANG-(1-7) as the sensitivity (pD2 values) did not change. The molecular mechanisms of ANG-(1-7) were first demonstrated by Sampaio et al. (40) in endothelial cells. Previously, Santos et al. (48) discovered that the Mas receptor was a binding site of ANG-(1-7). Activation by ANG-(1-7) causes dimerization of Mas receptors and the release of NO via activation of the AKT/eNOS pathway (40). In our study, we report that HS augments ANG-(1-7) signaling (ex vivo and in vivo) through activation of the Mas/AKT/eNOS pathway as both Mas receptor inhibitor (A-779) and eNOS inhibitor (L-NAME) prevent the effects of HS.
We also observed reduced eNOS expression in HFD-fed rats, and HS prevented this effect. Increased DNA methylation marks on the promoter of eNOS have been shown in vascular smooth muscle cells, which do not express eNOS (31). Interestingly, we provide evidence that HFD-fed rats had increased levels of DNMT1 in the aorta and that HS could prevent this increase. This definitely points out that eNOS expression might also be downregulated by DNA methylation. However, further studies are required to warrant any conclusion.
The role of ANG-(1-7) has been studied in various disorders such as hypertension, insulin resistance, and atherosclerosis (16). Like ACE inhibitors or AT1 receptor blockers, ANG-(1-7) attenuates the characteristics of metabolic syndrome (49). Oral formulation of ANG-(1-7) improves metabolism, and decreases the proinflammatory profile and fat deposition in HFD-fed mice as well as hypertensive rats (50,51). Prolonged ANG-(1-7) treatment has also been shown to improve endothelial dysfunction and oxidative stress in a mouse model of obesity (52). Recently, the therapeutic potential of ANG-(1-7) axis was highlighted by the development of an orally active nonpeptide Mas agonist, such as AVE 0991 and CGEN-865S. AVE 0991 showed beneficial effects in heart dysfunction, atherosclerosis, blood pressure, and pulmonary remodeling (53–56). These observations further confirm the protective role of ANG-(1-7) in different models of cardiovascular disorders.
Either Mas deficiency or Mas inhibition has been shown to ablate ANG-(1-7)–induced vasodilator responses or NO availability (40,48). Here, we report that mRNA levels of the Mas receptor gene decreases insulin resistance, and this may explain the impaired vasodilator responses to ANG-(1-7). Interestingly, HS treatment increases the expression of Mas receptor, and hence ANG-(1-7) signaling. This raises an interesting question of whether HSP72 interacts directly or indirectly with Mas receptor to regulate vascular tone. Additional studies are needed to elucidate this interaction. Enhancement in ANG-(1-7) signaling can also be explained by the observed increase in the expression of ACE2 by HS. ACE2 is a major enzyme for ANG-(1-7) formation. Studies involving the activation of ACE2 further confirm the role of ANG-(1-7) axis cardiovascular dysfunction. Either pharmacological or genetic activation of ACE2 improved endothelial migration, and impaired tube formation in renin transgenic mice and endothelial dysfunction in SHR rats (57,58). ACE2 activation has been reported to enhance the reparative function of endothelial progenitor cells in the diabetic condition (59). Further, ACE2 overexpression has been shown to prevent atherosclerosis, cardiac fibrosis, and hypertrophy, and these effects were in part mediated through upregulation of ANG-(1-7) (60,61).
Endothelial dysfunction is a hallmark of cardiovascular risk factors and has been linked to pathophysiology of hypertension. We and others have shown (1,4) an opposite correlation between NO bioavailability and endothelial dysfunction. Hypertensive subjects generated higher levels of free radicals, which quench NO and thereby reduce its bioavailability (62). Exercise or other nonpharmacological approaches such as endurance training is a systemic way to increase NO levels and treat hypertension (63). Like exercise, we also provide evidence that HS significantly increases NO levels and reduces systolic blood pressure in insulin-resistant rats.
How Do SIRT1 and AMPK Mediate the Effects of HSP72 Induction?
SIRT1 (a silent mating type information regulator 2 homolog) is known to extend the life span and insulin sensitivity in calorie restriction (64). SIRT1 is also considered to be beneficial in endothelial biology. Previously, it was shown that overexpression of SIRT1 prevented oxidative stress–induced endothelial senescence and enhanced endothelial-dependent vasodilation by deacetylating eNOS (65,66). SIRT1 closely works with AMPK in the regulation of cell stress and energy homeostasis. The lipid metabolism in hepatocytes has been shown to be regulated by SIRT1 through AMPK (67). However, in skeletal muscles AMPK modulates SIRT1 by regulating the activity of enzymes (Nampt) in a salvage pathway (68). A recent report suggests that shear stress primes AMPK and SIRT1 to act synergistically to increase NO bioavailability in endothelial cells (69). In light of this evidence, we report that insulin resistance represents a state of vascular SIRT1 and p-AMPK deficiency, which may contribute to the endothelial dysfunction. The induction of HSP72 restores the expression of SIRT1 and p-AMPK, which is another novel finding of our study. Assuming that effects mediated by HSP72 induction are, in part, mediated by SIRT1, we used NAM to inhibit SIRT1. SIRT1 inhibition suppressed the ANG-(1-7)–induced relaxant responses and endothelial-dependent vasodilation. This was also associated with reduced p-eNOS, p-AMPK, and SIRT1 expression, indicating a direct role of SIRT1 in mediating the effects of HSP72.
In conclusion, we show that the elevation of HSP72 levels by HS treatment augments ANG-(1-7) signaling and protects against endothelial dysfunction in insulin resistance. This protection was tightly associated with enhanced ANG-(1-7)–induced eNOS phosphorylation. The increased DNMT1 expression in insulin resistance suggests an epigenetic regulation of eNOS and SIRT1 expression. We propose that the induction of HSP72 can be a realistic approach to amplify ANG-(1-7)/Mas/ACE2 signaling and to prevent hypertension (Fig. 8).
Article Information
Acknowledgments. The authors thank Sandeep Kumar and Jasmine Kaur for insightful discussion of this manuscript.
Funding. This research was supported by the Department of Biotechnolgy, Government of India (via. sanction no. BT/ PR13221/MED/12/448/2009), National Institute of Pharmaceutical Education and Research, which provided necessary facilities and funding.
Duality of Interest. No potential conflicts of interest relevant to this article were reported.
Author Contributions. P.A.K. designed and performed all the experiments, and wrote the manuscript. K.T. designed and supervised the experiments, and approved the final version of the manuscript. K.T. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1267/-/DC1.
- Received August 20, 2013.
- Accepted November 15, 2013.
- © 2014 by the American Diabetes Association.
Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.


text:
Canadian backcountry skiers Andy and Mike Traslin love skiing – whatever the conditions. As a result they have skied every single month for 104 months! We caught up with Andy to talk about their never-ending ski season.
How did your endless ski season get started?
Our original goal was to try to ski for 12 months in a row, one consecutive year. After that it just became part of my life. When we got to 85 I started thinking that it would be a good goal, a cool milestone, to hit 100.
How has it been possible to accomplish this? Have you travelled a lot?
What makes this possible is where we live in the Southwestern part of British Columbia. Here a deep snowpack typically tends to stick all year round in the high alpine. From our base it is two hours to Whistler and two hours to Mt. Baker.
I really enjoy traveling and seeing new areas. BC has endless terrain for backcountry skiing.
My favorite spot is Mt. Baker in Washington, USA. Mt Baker is a volcano that rises to 3,286 meters (10,770 feet), offers over 2,000 meters (7000 feet) of descent and glaciated terrain that has snow all year round. There's everything from short early winter powder laps to epic steep ski descents on the North Face in spring and summer.
Andy Traslin enjoying the endless winter.
Has there been any nearly missed months along the way?
As in any sport injuries happen. Mike had a knee injury. I pulled my back. But we still managed to get out there. Lately we have made sure to go out at the beginning of the month to leave more time in case injuries or unforeseen circumstances.
Have you noticed any significant changes in the snow cover during these years?
The past two years has been significantly less snowfall. Especially this year has been one of the worst seasons I can remember. Hopefully this is not a sign of things to come. I have grown up in this area and there were seasons like this in the 80s.
Have you skied together every month?
We ski together most of the summer months. But in the main part of the season it's good to get out and ski with other people. I also like to ski solo once in a while as well.
How would you describe yourself as a skier and mountaineer?
First and foremost I'm a skier but I also enjoy mountaineering. But when I discovered that I can climb up a mountain and ski back down I knew that was for me.
Andy Traslin hiking up last summer.
What motivates you to get out there and ski so much?
I just really like to be outside, especially in the mountains skiing. That is my passion. Turns all year have enabled me to be out there pushing myself to the limits and skiing all the time in all types of conditions.
I have a real passion for steep ski mountaineering. So sometimes you have take risks to reach your ultimate goal. The mountain has the last say. So you have to follow your intuition and rely on years of experience in the mountains and know when to turn around.
The moments that really stick with me are when the sun, stability and people conditions line up perfectly. All the planning and hard work line up for that moment. That feeling of being alive and one with nature and flowing with gravity in a natural way.
Watch "95 'Till Infinity – Traslin's Never Ending Shred"

subset: commoncrawl


text:
Sleep(Suspend) When Idle
From MythTV Official Wiki
Revision as of 07:27, 14 September 2012 by Wagnerrp (talk | contribs) (category specifically for python scripts using the python bindings)
The script below can be used to put a frontend to sleep whenever xscreensaver turns the display 'Off' via DPMS. It was written with Mythbuntu 12.04/XFCE in mind, however it could probably be adapted for others distros.
Every 30 seconds it checks if mythfrontend is running and no additional users are logged in (via SSH), if so, it will check the DPMS status via xset, and if it is 'Off' it will suspend the machine.
Make the script executable by your frontend user account, and configure it to run in the session start.
NOTE: DPMS has 4 states; on, sleep, suspend, and off. The script will only trigger on the 'Off' state.
#!/usr/bin/env python from subprocess import call, check_output import time # Mythtv frontend process name fecommand = "mythfrontend.real" # Command to use to suspend the computer sleepcmd= ('dbus-send --system --print-reply ' '--dest="org.freedesktop.UPower" ' '/org/freedesktop/UPower ' 'org.freedesktop.UPower.Suspend') # Start infinate loop while True : # Wait 30 seconds before performing checks time.sleep (30) # Determine if the Frontend is stopped festopped = call("pidof " + fecommand + " >nul", shell=True) # Get the number of logged in users numusers = check_output("who -q | grep users | awk -F'=' '{print $2}'", shell=True) # If the number of logged in users <2 and the front end is not stopped if int(numusers) < 2 and not festopped: # Get the monitor DPMS status status = check_output('xset q |grep "Monitor is "', shell=True) # If the monitor DMPS status is 'Off' if 'Off' in status: # Run the sleep command call(sleepcmd, shell=True)


text:
Learning Index >> Hand-blockprinted Textiles: Phyllis Barron and Dorothy Larcher
Barron and Larcher's order book for April 1938 to September 1944 is a clear illustration of the fall in demand for their textiles during this period, largely caused by the outbreak of the Second World War. Orders from April 1938 up to the end of 1939 occupy 156 of the 220 pages, whilst those for 1940-44 are condensed into the remainder. Prices shown range from a few shillings per yard to several guineas, depending on the material used and the number of times it was printed. Quantities for orders varied from a headscarf to considerable yardage for curtaining and upholstery.
Orders taken direct are mostly written in Phyllis Barron's hand and range from nearby good friends like Eve Simmonds or Robin Tanner, to titled patrons such as Lady Adare and Lady Russell. (No. 1, 2, 3, 4) Several figures from the contemporary craft world are also included, like the furniture maker, Eric Sharpe, or the wife of the potter Michael Cardew. (No. 5, 6)


text:
The Economist Audio Edition March 30th - April 5th 2013
English | Audio MP3 / 128 Kbps | 6 hrs 45 mins | 162 MB
English | Audio MP3 / 128 Kbps | 6 hrs 45 mins | 162 MB
The audio edition contains word-for-word recordings of all articles published in The Economist, read by professional broadcasters and actors. It is ideal for anyone who wants to listen to articles while travelling, exercising or just relaxing.
The Economist is an English-language weekly news and international affairs publication owned by "The Economist Newspaper Ltd" and edited in London. It has been in continuous publication since James Wilson established it in September 1843. As of summer 2007, its average circulation topped 1.2 million copies a week, about half of which are sold in North America. Consequently it is often seen as a transatlantic (as opposed to solely British) news source.
The aim of The Economist is "to take part in a severe contest between intelligence, which presses forward, and an unworthy, timid ignorance obstructing our progress."Subjects covered include international news, economics, politics, business, finance, science, technology, and the arts. The publication is targeted at the high-end "prestige" segment of the market and counts among its audience influential business and government decision-makers.
It takes a strongly argued editorial stance on many issues, especially its support for free trade and fiscal conservatism; it can thus be considered as a magazine which practises advocacy journalism.
Although The Economist calls itself a newspaper and refers to its staff as correspondents, it is printed in magazine form on glossy paper, like a newsmagazine.
The Economist belongs to The Economist Group. The publication interests of the group include the CFO brand family as well as the annual World in…, the lifestyle quarterly Intelligent Life, European Voice and Roll Call (known as "the Newspaper of Capitol Hill"). Another part of the group is The Economist Intelligence Unit, a research and advisory company providing country, industry and management analysis worldwide. Since 1928, half the shares of The Economist Group have been owned by the Financial Times, a subsidiary of Pearson PLC, and the other half by a group of independent shareholders, including many members of the staff. The editor's independence is guaranteed by the existence of a board of trustees, which formally appoints him and without whose permission he cannot be removed.


text:
Located right next door to trendy, bustling Maroochydore on the Sunshine Coast, Cotton Tree is fringed by the sandy banks of the Maroochy River to the north and the white silica beaches of the blue Pacific Ocean to the west.
Surrounded by so much clean, pristine water, it's only natural that Cotton Tree is home to an impressive range of watersport activities. Hire a boat and head to Pincushion Island at the mouth of the river. This tall sand and rock formation is famous with the locals for its fishing and confounds visitors as it appears to move position with the changing tide.
The walk from Cotton Tree to Alexandra Headland takes in glorious, sweeping ocean views and along the way you'll find idyllic spots to picnic and breathe in the fresh ocean air.
On the weekends, Cotton Tree comes alive with Sunday markets and a thriving al fresco cafe scene. Browse through the stalls and sample some of the fresh local produce on offer. For funky fashion boutiques or stylish homewares head to trendy Duporth Avenue and shop til you drop!
Soaking up the tranquil river views over a long lazy lunch in one of the many fine riverfront restaurants completes a perfect day at beautiful Cotton Tree.
Note: Information on listed products and services are provided by the operator and were correct at the time of publishing. Rates are indicative based on the minimum and maximum available prices of products and services. Please visit the operator's website for further information. All prices quoted are in Australian dollars (AUD).


text:
Kalff (Calff, Kalf) family
Kalff (Calff, Kalf) is a Dutch Mennonite family of Zaandam, province of North Holland, which played an important part in the economic prosperity of this town. They were merchants and shipowners, often engaging in whaling as far away as Greenland. Cornelis Michielsz Calff (1652-1721), who was a shipbuilder and equipped a number of boats for his own business in whaling grounds near Greenland, and who was consulted by Peter the Great of Russia when he visited Zaandam in 1717, was a preacher of the Flemish congregation of Zaandam from 1680, and of the United Flemish and Waterlander Nieuwe Huys congregation from 1687 till his death in 1721. His son Nicolaas Kalf (Claes Cornelisz Calf, 1677-1734), who was a wholesale dealer, shipowner, and manufacturer of Zaandam, and was known for his excellent collection of coins and other art treasures, was a promoter of the founding of the West Zaandam Mennonite orphanage in 1713-1714. J. Kalff, a lawyer, was one of the founders of the congregation at The Hague in 1880-1881.
Doopsgezinde Bijdragen (1896): 64, 69 f.
Lootsma, S. Het Nieuwe Huys, 1687-1937. Zaandam, 1937: 31, 35 f., 48, 65, 115 f., 141, 186.
|Nanne van der Zijpp
|Date Published
Cite This Article
MLA style
Zijpp, Nanne van der. "Kalff (Calff, Kalf) family." Global Anabaptist Mennonite Encyclopedia Online. 1957. Web. 4 Aug 2020. https://gameo.org/index.php?title=Kalff_(Calff,_Kalf)_family&oldid=120816.
APA style
Zijpp, Nanne van der. (1957). Kalff (Calff, Kalf) family. Global Anabaptist Mennonite Encyclopedia Online. Retrieved 4 August 2020, from https://gameo.org/index.php?title=Kalff_(Calff,_Kalf)_family&oldid=120816.
Adapted by permission of Herald Press, Harrisonburg, Virginia, from Mennonite Encyclopedia, Vol. 3, p. 138. All rights reserved.
©1996-2020 by the Global Anabaptist Mennonite Encyclopedia Online. All rights reserved.


text:
How ARPA-e is "Winning the Future"
You are here
PRESIDENT BARACK OBAMA: Two years ago, I said that we needed to reach a level of research and development we haven't seen since the height of the space race. We'll fund the Apollo projects of our time.
Okay, here we go. A big one. Off the ground – (inaudible).
Just as the old DARPA funded ARPANET, which later became the basis of the Internet and the development of the global position satellite system. Those were truly game-changers in the way information is generated throughout the world. We need to do the same for energy.
ARUN MAJUMDAR: My name is Arun Majumdar. I'm the director of Advanced Research Projects Agency for Energy, or ARPA-E, of the Department of Energy.
We have to realize that we are in a global competition. All the technologies that are required for global, sustainable economic growth do not exist today. There are many economies that are growing faster than us and they're all looking to do it in a sustainable way.
Because the program managers and the people in the ARPA-E program are so good – top scientists and engineers in their own right – they can sort through and identify really new ideas.
There are many reports of how fast we have gone, and that is extremely important because if you are asking our PIs and proposers and our tech community, entrepreneurs, to increase their pace, they should ask the government to be fast.
MR. : Technically, it's a new clean-energy revolution, both in the way we use energy much more efficiently but also the way we generate energy. We are the technological leaders and we will prosper.
MR. : We have just found out that the amount of investment that we made in the first round, which is $150 million – the private sector has seen the de-risking and the progress that these technologies have made. Okay, not all of them, but a few of them and the amount of private-sector dollars that have come in has actually exceeded $150 million.
MR. : This is the biggest business opportunity that the United States has. If we develop these technologies, make them locally, sell them globally, that is the answer for our economic prosperity as well as our national security.
PRESIDENT OBAMA: We know what it takes to compete for the jobs and industries of our time. We need to out-innovate, out-educate and out-build the rest of the world. (Applause.)


text:
Porsche vs Lamborghini & Rally Driving Taster
Taster Experience
Valid For 10 Months
Extra Laps Available
Choice of Location
This voucher is for a taster session driving a Porsche Cayman, Lamborghini Gallardo and Subaru WRX STI.
If you know someone who would want to drive pit these three cars against ear other, then this is the ideal gift.
This voucher is valid for 10 months only from the date of purchase and therefore the day of the experience must be before this date.
Extra laps can be added to this experience to extend the driving time or you can choose a Porsche vs Lamborghini & Rally Driving Experience or a Porsche vs Lamborghini & Rally Extended Driving Experience voucher.
- Welcome and registration
- Choice of location
- Taster session driving a Porsche Cayman
- Taster session driving a Lamborghini Gallardo
- Taster session driving a Subaru WRX STI
- Collection of your driving certificate
Stafford Driving Centre – 3 Laps
Stafford Driving Centre, Clanford Road, Seighford, Stafford. ST18 9PF
Anglesey Race Circuit – 2 laps
Anglesey Race Circuit, Ty Croes, Anglesey, Wales. LL63 5TF
Three Sisters Circuit – 3 Laps
Three Sisters Circuit, Three Sisters Road, Ashton-in-Makerfield, Wigan. WN4 8DD
Tockwith Motorsports Centre – 3 Laps
Tockwith Motorsport Centre, Roman Road/ Rudgate Lane, Tockwith, York. YO26 7DW
More Info
Please allow approximately 2 hours at the venue for a Porsche vs Lamborghini & Rally Driving Taster.
You must hold a full manual driving licence, be less than 6'6" and weigh less than 18 ½ stone.
Events run at Stafford on selected dates inc weekends between January & December.
Events run at Anglesey on selected dates inc weekends from March to October.
Events run at Three Sisters on selected Mondays from March to September.
Events run at Tockwith on selected dates inc weekends between March & October.
Bookings made between 11am and 3pm are classed as premium time slots and will incur an additional cost of £10. These are subject to availability, please enquire at time of booking.
Optional Policies:
Cancellation Policy – To cover you in case your event needs to be rescheduled.
Damage Waiver Policy – To cover the driver in the event of an accident.
These can be found and purchased at checkout.
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