Impaired Albumin Uptake and Processing Promote Albuminuria in OVE26 Diabetic Mice.

The importance of proximal tubules dysfunction to diabetic albuminuria is uncertain. OVE26 mice have the most severe albuminuria of all diabetic mouse models but it is not known if impaired tubule uptake and processing are contributing factors. In the current study fluorescent albumin was used to follow the fate of albumin in OVE26 and normal mice. Compared to normal urine, OVE26 urine contained at least 23 times more intact fluorescent albumin but only 3-fold more 70 kD fluorescent dextran. This indicated that a function other than size selective glomerular sieving contributed to OVE26 albuminuria. Imaging of albumin was similar in normal and diabetic tubules for 3 hrs after injection. However 3 days after injection a subset of OVE26 tubules retained strong albumin fluorescence, which was never observed in normal mice. OVE26 tubules with prolonged retention of injected albumin lost the capacity to take up albumin and there was a significant correlation between tubules unable to eliminate fluorescent albumin and total albuminuria. TUNEL staining revealed a 76-fold increase in cell death in OVE26 tubules that retained fluorescent albumin. These results indicate that failure to process and dispose of internalized albumin leads to impaired albumin uptake, increased albuminuria, and tubule cell apoptosis.

Nutritional treatment for acute and chronic traumatic brain injury patients.

Proper nutrition is critical for recovery from traumatic brain injury (TBI). Prompt enteral feeding of moderate to severe TBI patients has been associated with significantly lower mortality and rates of infection. Probiotic supplementation has been associated with significantly lower rates of infection in TBI and other trauma patients. Human studies have suggested that supplementation with omega 3 fats, vitamin D, N-Acetylcysteine, branched chain amino acids, and zinc may be helpful for recovery from TBI. Animal TBI models have suggested that alpha-lipoic acid, magnesium, taurine, coenzyme Q10, and many phytonutrients (such as resveratrol) are also helpful. Unfortunately, recent human clinical trials with citicoline in TBI and stroke patients have produced disappointing results. Much more research is needed on multifaceted nutritional strategies to treat TBI patients in both the immediate post-injury phase and throughout the patients lifespan.

Impaired baroreflex control of renal sympathetic nerve activity in type 1 diabetic mice (OVE26).

To investigate the effects of chronic diabetes on baroreflex control of renal sympathetic nerve activity (RSNA), OVE26 diabetic (transgenic mouse line which develops hyperglycemia within the first 3 weeks after birth) and FVB control mice 5-6 months old were studied. Under anesthesia, RSNA in response to sodium nitroprusside (SNP)- and phenylephrine (PE)-induced mean arterial pressure changes (DeltaMAP) were measured. Baroreflex-induced inhibition of RSNA during PE infusion was characterized using the sigmoid logistic function curve. Baroreflex-induced excitation of RSNA during SNP infusion was characterized by the RSNA vs. DeltaMAP relationship. Mean arterial pressure (MAP) responses to the left aortic depressor nerve (ADN) stimulation were evaluated. Compared to FVB control, we found in OVE26 mice that (1) RSNA in response to MAP increase during PE infusion was dramatically reduced, as characterized by the maximal gain of the RSNA sigmoid logistic function curve (FVB: -20.0+/-5.1; OVE26: -7.6+/-0.8%/mm Hg, P<0.05); (2) RSNA in response to MAP decrease during SNP infusion was also attenuated (P<0.05); (3) MAP responses to ADN stimulation were reduced (P<0.05). We concluded that chronic diabetes impairs baroreflex control of RSNA in OVE26 diabetic mice. The use of the transgenic OVE26 diabetic mouse model may underlie a foundation for the further understanding of diabetes-induced autonomic neuropathy.

The role of pyruvate carboxylase in insulin secretion and proliferation in rat pancreatic beta cells.

AIMS/HYPOTHESIS: Pyruvate carboxylase (PC) or pyruvate dehydrogenase (PDH) is required to transfer carbons from pyruvate into the Krebs cycle. PC activity is preserved in the islets of obese animals, but it is reduced in the islets of animal models of type 2 diabetes, suggesting that PC is important in beta cell adaptation to insulin resistance and that PC reduction may lead to beta cell failure. METHODS: To confirm the significance of PC, we first lowered activity using Pc (now known as Pcx) small interfering RNA (siRNA) in INS-1 cells and in dispersed rat islet cells. Second, we overexpressed PC in INS-1 cells, and third, we inhibited PDH by overexpressing the gene encoding pyruvate dehydrogenase kinase 4 (Pdk4) in INS-1 cells. RESULTS: Treatment of INS-1 cells or dispersed rat islet cells with Pc siRNA resulted in a significant reduction in insulin secretion in both cell types and reduced proliferation in INS-1 cells. This treatment also reduced the content of oxaloacetate, malate and ATP, as well as the NADPH:NADP(+) ratio and activity of the pyruvate-malate shuttle. Overexpression of PC in INS-1 cells led to an elevation of insulin secretion and cell proliferation, whereas inhibition of PDH activity by overexpressing Pdk4 in INS-1 cells did not reduce insulin secretion. CONCLUSIONS/INTERPRETATION: Our findings indicate that the PC pathway in beta cells might play a key role in pyruvate metabolism, insulin secretion and cell proliferation.

Malic enzyme is present in mouse islets and modulates insulin secretion.

AIMS/HYPOTHESIS: The pyruvate-malate shuttle is a metabolic cycle in pancreatic beta cells and is important for beta cell function. Cytosolic malic enzyme (ME) carries out an essential step in the shuttle by converting malate to pyruvate and generating NADPH. In rat islets the pyruvate-malate shuttle may regulate insulin secretion and it has been shown to play a critical role in adaptation to obesity and insulin resistance. However, ME has not been demonstrated in mouse islets and three reports indicate that mouse islets contain no ME activity. If mouse islets lack ME, rat and mouse islets must regulate insulin secretion by different mechanisms. METHODS: We measured ME activity by a fluorometric enzymatic assay and Me mRNA by real-time PCR. ME activity was also measured in streptozotocin-treated mouse islets. FACS-purified beta cells were obtained from MIP-GFP mouse islets, agouti-L obese mouse islets and mouse beta cell line MIN-6. Insulin secretion and NADPH/NADP(+) ratios were measured in Me siRNA-treated beta cells. RESULTS: ME activity and Me mRNA were present in C57BL/6 mouse islets. ME activity was reduced in streptozotocin-treated mouse islets. ME activity was also measurable in FACS-purified mouse beta cells. In addition, ME activity was significantly increased in obese agouti-L mouse islets and the mouse MIN-6 cell line. Me siRNA inhibited ME activity and reduced glucose-stimulated insulin secretion and also inhibited NADPH products. CONCLUSIONS/INTERPRETATION: Mouse islets contain ME, which plays a significant role in regulating insulin secretion.

Functional changes in baroreceptor afferent, central and efferent components of the baroreflex circuitry in type 1 diabetic mice (OVE26).

Baroreflex control of heart rate (HR) is impaired in diabetes mellitus. We hypothesized that diabetes mellitus induced functional changes of neural components at multiple sites within the baroreflex arc. Type 1 diabetic mice (OVE26) and FVB control mice were anesthetized. Baroreflex-mediated HR responses to sodium nitroprusside- (SNP) and phenylephrine- (PE) induced mean arterial blood pressure (MAP) changes were measured. Baroreceptor function was characterized by measuring the percent (%) change of baseline integrated aortic depressor nerve activity (Int ADNA) in response to SNP- and PE-induced MAP changes. The HR responses to electrical stimulation of the left aortic depressor nerve (ADN) and the right vagus nerve were assessed. Compared with FVB control mice, we found in OVE26 mice that (1) baroreflex-mediated bradycardia and tachycardia were significantly reduced. (2) The baroreceptor afferent function in response to MAP increase did not differ, as assessed by the parameters of the logistic function curve. But, the inhibition of Int ADNA in response to MAP decrease was significantly attenuated. (3) The maximum amplitude of bradycardic responses to right vagal efferent stimulation was augmented. (4) In contrast, the maximum amplitude of bradycardic responses to left ADN stimulation was decreased. Since Int ADNA was preserved in response to MAP increase and HR responses to vagal efferent stimulation were augmented, we conclude that a deficit of the central mediation of baroreflex HR contributes to the overall attenuation of baroreflex sensitivity in OVE26 mice. The successful conduction of physiological experiments on the ADN in OVE26 mice may provide a foundation for the understanding of cellular and molecular mechanisms of diabetes-induced cardiac neuropathy.

Morphology and topography of nucleus ambiguus projections to cardiac ganglia in rats and mice.

Vagal efferent axons from the nucleus ambiguus (NA) innervate ganglionated plexuses in the dorsal surface of cardiac atria, which in turn, may have different functional roles in cardiac regulation. However, the morphology and topography of vagal efferent projections to these ganglionated plexuses in rats and mice have not been well delineated. In the present study, we injected the tracer 1,1'-dioctadecyl-3,3,3',3' tetramethylindocarbocyanine methanesulfonate (DiI) into the left NA to label vagal efferent axons and terminals in cardiac ganglia and administered Fluoro-Gold (FG) i.p. to stain cardiac ganglia. Then, we used confocal microscopy and a Neurolucida 3-D Digitization System to qualitatively and quantitatively examine the distribution and structure of cardiac ganglia, and NA efferent projections to cardiac ganglia in the whole-mounts of Sprague-Dawley (SD) rats and FVB mice. Our observations were: 1) Cardiac ganglia of different shapes and sizes were distributed in the sinoatrial (SA) node, atrioventricular (AV) node, and lower pulmonary vein (LPV) regions on the dorsal surface of the atria. In each region, several ganglia formed a ganglionated plexus. The plexuses at different locations were interconnected by nerves. 2) Vagal efferent fibers ramified within cardiac ganglia, formed a complex network of axons, and innervated cardiac ganglia with very dense basket endings around individual cardiac principal neurons (PNs). 3) The percent of the PNs in cardiac ganglia which were innervated by DiI-labeled axons was 54.3+/-3.2% in mice vs. 53.2+/-3.2% in rats (P>0.10). 4) The density of axonal putative-synaptic varicosities on the surface of PNs was 0.15+/-0.02/microm(2) in mice vs. 0.16+/-0.02/microm(2) in rats (P>0.10). Thus, the distributions of cardiac ganglia and vagal efferent projections to cardiac ganglia in mice and rats were quite similar both qualitatively and quantitatively. Our study provides the structural foundation for future investigation of functional differentiation of ganglionated plexuses and the brain-heart circuitry in rodent models of human disease.

Viewing the functional consequences of traumatic brain injury by using brain SPECT.

High-resolution brain SPECT is increasingly benefiting from improved image processing software and multiple complementary display capabilities. This enables detailed functional mapping of the disturbances in relative perfusion occurring after TBI. The patient population consisted of 26 cases (ages 8-61 years)between 3 months and 6 years after traumatic brain injury.A very strong case can be made for the routine use of Brain SPECT in TBI. Indeed it can provide a detailed evaluation of multiple functional consequences after TBI and is thus capable of supplementing the clinical evaluation and tailoring the therapeutic strategies needed. In so doing it also provides significant additional information beyond that available from MRI/CT. The critical factor for Brain SPECT's clinical relevance is a carefully designed technical protocol, including displays which should enable a comprehensive description of the patterns found, in a user friendly mode.

Metallothionein alleviates cardiac contractile dysfunction induced by insulin resistance: role of Akt phosphorylation, PTB1B, PPARgamma and c-Jun.

AIMS/HYPOTHESIS: Insulin resistance is concomitant with metabolic syndrome, oxidative stress and cardiac contractile dysfunction. However, the causal relationship between oxidative stress and cardiac dysfunction is unknown. This study was designed to determine the impact of overexpression of the cardiac antioxidant metallothionein on cardiac dysfunction induced by insulin resistance in mice. METHODS: Whole-body insulin resistance was generated in wild-type FVB and metallothionein transgenic mice by feeding them with sucrose for 12 weeks. Contractile and intracellular Ca(2+) properties were evaluated in ventricular myocytes using an IonOptix system. The contractile indices analysed included: peak shortening (PS), time to 90% PS (TPS(90)), time to 90% relengthening (TR(90)), half-width duration, maximal velocity of shortening (+dL/dt) and relengthening (-dL/dt), fura-fluorescence intensity change (DeltaFFI) and decay rate (tau). RESULTS: The sucrose-fed mice displayed glucose intolerance, enhanced oxidative stress, hyperinsulinaemia, hypertriglyceridaemia and normal body weight. Compared with myocytes in starch-fed mice, those from sucrose-fed mice exhibited depressed PS, +dL/dt, -dL/dt, prolonged TR(90) and decay rate, and reduced DeltaFFI associated with normal TPS(90) and half-width duration. Western blot analysis revealed enhanced basal, but blunted insulin (15 mU/g)-stimulated Akt phosphorylation. It also showed elevated expression of insulin receptor beta, insulin receptor tyrosine phosphorylation, peroxisome proliferator-activated receptor gamma, protein tyrosine phosphatase 1B and phosphorylation of the transcription factor c-Jun, associated with a reduced fold increase of insulin-stimulated insulin receptor tyrosine phosphorylation in sucrose-fed mice. All western blot findings may be attenuated or ablated by metallothionein. CONCLUSIONS/INTERPRETATION: These data indicate that oxidative stress may play an important role in cardiac contractile dysfunction associated with glucose intolerance and possibly related to alteration in insulin signalling at the receptor and post-receptor levels.

Long-term effect of maternal obesity on pancreatic beta cells of offspring: reduced beta cell adaptation to high glucose and high-fat diet challenges in adult female mouse offspring.

AIM/HYPOTHESIS: Obesity is a global problem with high risks of cardiovascular diseases, stroke and type 2 diabetes. It is well known that maternal obesity affects offspring by inducing malformation, functional abnormalities in many organs and cells, and by increased risk of obesity and type 2 diabetes. However, little is known about abnormalities induced by maternal obesity in pancreatic beta cells of offspring. METHODS: We used mouse mothers with the Agouti yellow modification on a C57BL/6 background as a maternal model of normoglycaemic obesity, and produced Agouti-negative offspring. Half of the offspring were fed a high-fat diet. Offspring glucose tolerance was tested at different ages, and animals were killed at 50 weeks of age for islet function analysis. RESULTS: Maternal obesity impaired glucose tolerance in female offspring fed a high-fat diet, and significantly reduced insulin secretion at 50 weeks of age in female offspring that had been fed a normal diet and high-fat diet. Insulin secretion and glucose potentiation from these islets were significantly reduced. Islet protein, DNA and insulin contents were increased while glyceraldehyde-3-phosphate dehydrogenase and transketolase activities were reduced in female offspring. CONCLUSIONS/INTERPRETATION: Our results indicate that maternal obesity has a long-term effect on the beta cells of female, but not of male, offspring, and leads to increased risk of gestational diabetes and type 2 diabetes in the offspring's later lives.

Enhanced rat beta-cell proliferation in 60% pancreatectomized islets by increased glucose metabolic flux through pyruvate carboxylase pathway.

Islet beta-cell proliferation is a very important component of beta-cell adaptation to insulin resistance and prevention of type 2 diabetes mellitus. However, we know little about the mechanisms of beta-cell proliferation. We now investigate the relationship between pyruvate carboxylase (PC) pathway activity and islet cell proliferation 5 days after 60% pancreatectomy (Px). Islet cell number, protein, and DNA content, indicators of beta-cell proliferation, were increased two- to threefold 5 days after Px. PC and pyruvate dehydrogenase (PDH) activities increased only approximately 1.3-fold; however, islet pyruvate content and malate release from isolated islet mitochondria were approximately threefold increased in Px islets. The latter is an indicator of pyruvate-malate cycle activity, indicating that most of the increased pyruvate was converted to oxaloacetate (OAA) through the PC pathway. The contents of OAA and malate, intermediates of the pyruvate-malate cycle, were also increased threefold. PDH and citrate content were only slightly increased. Importantly, the changes in cell proliferation parameters, glucose utilization, and oxidation and malate release were partially blocked by in vivo treatment with the PC inhibitor phenylacetic acid. Our results suggest that enhanced PC pathway in Px islets may have an important role in islet cell proliferation.

Expression of cGMP-specific phosphodiesterase 9A mRNA in the rat brain.

cGMP has been implicated in the regulation of many essential functions in the brain, such as synaptic plasticity, phototransduction, olfaction, and behavioral state. Cyclic nucleotide phosphodiesterase (PDE) hydrolysis of cGMP is the major mechanism underlying the clearance of cGMP and is likely to be important in any process that depends on intracellular cGMP. PDE9A has the highest affinity for cGMP of any PDE, and here we studied the localization of this enzyme in the rat brain using in situ hybridization. PDE9A mRNA is widely distributed throughout the brain with varying regional expression. The pattern of PDE9A mRNA expression closely resembles that of soluble guanylyl cyclase (sGC) in the rat brain, suggesting a possible functional association or coupling of these two enzymes in the regulation of cGMP levels. Most of the brain areas expressing PDE9A mRNA also contain neuronal nitric oxide synthase (NOS), the enzymatic source of NO and the principal activator of sGC. PDE9A is the only cGMP-specific PDE with significant expression in the forebrain, and as such is likely to play an important role in NO-cGMP signaling.

The precautionary principle in environmental science.

Environmental scientists play a key role in society's responses to environmental problems, and many of the studies they perform are intended ultimately to affect policy. The precautionary principle, proposed as a new guideline in environmental decision making, has four central components: taking preventive action in the face of uncertainty; shifting the burden of proof to the proponents of an activity; exploring a wide range of alternatives to possibly harmful actions; and increasing public participation in decision making. In this paper we examine the implications of the precautionary principle for environmental scientists, whose work often involves studying highly complex, poorly understood systems, while at the same time facing conflicting pressures from those who seek to balance economic growth and environmental protection. In this complicated and contested terrain, it is useful to examine the methodologies of science and to consider ways that, without compromising integrity and objectivity, research can be more or less helpful to those who would act with precaution. We argue that a shift to more precautionary policies creates opportunities and challenges for scientists to think differently about the ways they conduct studies and communicate results. There is a complicated feedback relation between the discoveries of science and the setting of policy. While maintaining their objectivity and focus on understanding the world, environmental scientists should be aware of the policy uses of their work and of their social responsibility to do science that protects human health and the environment. The precautionary principle highlights this tight, challenging linkage between science and policy.

Characterization of contractile function in diabetic hypertensive cardiomyopathy in adult rat ventricular myocytes.

Diabetes and hypertension both produce myocardial dysfunction that accelerates cardiovascular morbidity and mortality. Coexistence of the two often results in a more severe cardiomyopathy than either process alone. The purpose of this study was to characterize the contractile function of diabetic hypertensive cardiomyopathy at the single myocyte level. Adult spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats were made diabetic with a single injection (55 mg/kg) of streptozotocin (STZ). Contractile properties of ventricular myocytes were evaluated, including peak shortening (PS), time-to-peak shortening (TPS), time-to-90% relengthening (TR90) and maximal velocities of shortening/relengthening (+/-dL/d t). The experimental animals exhibited enlarged heart size, elevated blood glucose and systolic blood pressure. PS was unchanged (SHR), enhanced (WKY-STZ) or depressed (SHR-STZ) compared to control (WKY). Myocytes from all experimental groups displayed prolonged TPS and TR90 compared to the WKY group, although only those from the hypertensive groups (SHR, SHR-STZ) were associated with reduced +/-dL/d t. Additionally, myocytes from the WKY-STZ but not the SHR or the SHR-STZ groups exhibited impaired responsiveness to increased extracellular Ca2+. Myocytes from the SHR-STZ group displayed a leftward shift of the stimulus frequency-peak shortening response curve compared to the WKY group. These results confirmed observations at the multicellular levels that combination of diabetes and hypertension results in a greater impairment of cardiac contractile function than is seen with either disease alone.

Climate change and emerging infectious diseases.

The ranges of infectious diseases and vectors are changing in altitude, along with shifts in plant communities and the retreat of alpine glaciers. Additionally, extreme weather events create conditions conducive to clusters of insect-, rodent- and water-borne diseases. Accelerating climate change carries profound threats for public health and society.

Marine swimming-related illness: implications for monitoring and environmental policy.

There is increasing evidence that environmental degradation may be contributing to an increase in marine-related diseases across a wide range of taxonomic groups. This includes a growing number of reports of both recreational and occupational users of marine waters developing gastrointestinal, respiratory, dermatologic, and ear, nose, and throat infections. The duration and type of exposure, concentration of pathogens, and host immunity determine the risk of infection. Public health authorities may not be able to accurately predict the risk of waterborne disease from marine waters due to the limitations of conventional monitoring, as well as erroneous perceptions of pathogen life span in marine systems. Pathogens undetectable by conventional methods may remain viable in marine waters, and both plankton and marine sediments may serve as reservoirs for pathogenic organisms, which can emerge to become infective when conditions are favorable. In this paper we address the environmental factors that may contribute to illness, the types of associated economic costs, the issues of water quality monitoring and the policy implications raised by the apparent rise in incidence of marine water-related illnesses.

Overexpression of metallothionein in pancreatic beta-cells reduces streptozotocin-induced DNA damage and diabetes.

The release of reactive oxygen species (ROS) has been proposed as a cause of streptozotocin (STZ)-induced beta-cell damage. This initiates a destructive cascade, consisting of DNA damage, excess activation of the DNA repair enzyme poly(ADP-ribose) polymerase, and depletion of cellular NAD+. Metallothionein (MT) is an inducible antioxidant protein that has been shown to protect DNA from chemical damage in several cell types. Therefore, we examined whether overexpression of MT could protect beta-cell DNA and thereby prevent STZ-induced diabetes. Two lines of transgenic mice were produced with up to a 30-fold elevation in beta-cell MT. Cultured islets from control mice and MT transgenic mice were exposed to STZ. MT was found to decrease STZ-induced islet disruption, DNA breakage, and depletion of NAD+. To assess in vivo protection, transgenic and control mice were injected with STZ. Transgenic mice had significantly reduced hyperglycemia. Ultrastructural examination of islets from STZ-treated mice showed that MT prevented degranulation and cell death. These results demonstrate that MT can reduce diabetes and confirm the DNA damage mechanism of STZ-induced beta-cell death.

West Nile virus and the climate.

West Nile virus is transmitted by urban-dwelling mosquitoes to birds and other animals, with occasional "spillover" to humans. While the means by which West Nile virus was introduced into the Americas in 1999 remain unknown, the climatic conditions that amplify diseases that cycle among urban mosquitoes, birds, and humans are warm winters and spring droughts. This information can be useful in generating early warning systems and mobilizing timely and the most environmentally friendly public health interventions. The extreme weather conditions accompanying long-term climate change may also be contributing to the spread of West Nile virus in the United States and Europe.

Climate variability and change in the United States: potential impacts on water- and foodborne diseases caused by microbiologic agents.

Exposure to waterborne and foodborne pathogens can occur via drinking water (associated with fecal contamination), seafood (due to natural microbial hazards, toxins, or wastewater disposal) or fresh produce (irrigated or processed with contaminated water). Weather influences the transport and dissemination of these microbial agents via rainfall and runoff and the survival and/or growth through such factors as temperature. Federal and state laws and regulatory programs protect much of the U.S. population from waterborne disease; however, if climate variability increases, current and future deficiencies in areas such as watershed protection, infrastructure, and storm drainage systems will probably increase the risk of contamination events. Knowledge about transport processes and the fate of microbial pollutants associated with rainfall and snowmelt is key to predicting risks from a change in weather variability. Although recent studies identified links between climate variability and occurrence of microbial agents in water, the relationships need further quantification in the context of other stresses. In the marine environment as well, there are few studies that adequately address the potential health effects of climate variability in combination with other stresses such as overfishing, introduced species, and rise in sea level. Advances in monitoring are necessary to enhance early-warning and prevention capabilities. Application of existing technologies, such as molecular fingerprinting to track contaminant sources or satellite remote sensing to detect coastal algal blooms, could be expanded. This assessment recommends incorporating a range of future scenarios of improvement plans for current deficiencies in the public health infrastructure to achieve more realistic risk assessments.