Edward C. Carlson

High-fat diet-induced juvenile obesity leads to cardiomyocyte dysfunction and upregulation of Foxo3a transcription factor independent of lipotoxicity and apoptosis.

Background Obesity is associated with dyslipidemia, which leads to elevated triglyceride and ceramide levels, apoptosis and compromised cardiac function. Methods To determine the role of high-fat diet-induced obesity on cardiomyocyte function, weanling male Sprague–Dawley rats were fed diets incorporating 10% of kcal or 45% of kcal from fat. Mechanical function of ventricular myocytes was evaluated including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR90) and maximal velocity of shortening and relengthening (± dl/dt). Intracellular Ca2+ properties were assessed using fluorescent microscopy. Results High-fat diet induced hyperinsulinemic insulin-resistant obesity with depressed PS, ± dl/dt, prolonged TPS/TR90 reduced intracellular Ca2+ release and Ca2+ clearing rate in the absence of hypertension, diabetes, lipotoxicity and apoptosis. Myocyte responsiveness to increased stimulus frequency and extracellular Ca2+ was compromised. SERCA2a and phospholamban levels were increased, whereas phosphorylated phospholamban and potassium channel (Kv1,2) were reduced in high-fat diet group. High-fat diet upregulated the forkhead transcription factor Foxo3a, and suppressed mitochondrial aconitase activity without affecting expression of the caloric sensitive gene silent information regulator 2 (Sir2), protein nitrotyrosine formation, lipid peroxidation and apoptosis. Levels of endothelial nitric oxide synthase (NOS), inducible NOS, triglycerides and ceramide were similar between the two groups. Conclusions Collectively, our data show that high-fat diet-induced obesity resulted in impaired cardiomyocyte function, upregulated Foxo3a transcription factor and mitochondrial damage without overt lipotoxicity or apoptosis.

Autophagy plays an essential role in the clearance of Pseudomonas aeruginosa by alveolar macrophages

Intracellular bacteria have been shown to cause autophagy, which impacts infectious outcomes, whereas extracellular bacteria have not been reported to activate autophagy. Here, we demonstrate that Pseudomonas aeruginosa, a Gram-negative extracellular bacterium, activates autophagy with considerably increased LC3 punctation in both an alveolar macrophage cell line (MH-S) and primary alveolar macrophages. Using the LC3 Gly120 mutant, we successfully demonstrated a hallmark of autophagy, conjugation of LC3 to phosphatidylethanolamine (PE). The accumulation of typical autophagosomes with double membranes was identified morphologically by transmission electron microscopy (TEM). Furthermore, the increase of PE-conjugated LC3 was indeed induced by infection rather than inhibition of lysosome degradation. P. aeruginosa induced autophagy through the classical beclin-1–Atg7–Atg5 pathway as determined by specific siRNA analysis. Rapamycin and IFN-γ (autophagy inducers) augmented bacterial clearance, whereas beclin-1 and Atg5 knockdown reduced intracellular bacteria. Thus, P. aeruginosa-induced autophagy represents a host protective mechanism, providing new insight into the pathogenesis of this infection.

Podocyte-Specific Overexpression of the Antioxidant Metallothionein Reduces Diabetic Nephropathy

Podocytes are critical components of the selective filtration barrier of the glomerulus and are susceptible to oxidative damage. For investigation of the role of oxidative stress and podocyte damage in diabetic nephropathy, transgenic mice that overexpress the antioxidant protein metallothionein (MT) specifically in podocytes (Nmt mice) were produced. MT expression was increased six- and 18-fold in glomeruli of two independent lines of Nmt mice, and podocyte-specific overexpression was confirmed. Glomerular morphology and urinary albumin excretion were normal in Nmt mice. OVE26 transgenic mice, a previously reported model of diabetic nephropathy, were crossed with Nmt mice to determine whether an antioxidant transgene targeted to podocytes could reduce diabetic nephropathy. Double-transgenic OVE26Nmt mice developed diabetes similar to OVE26 mice, but MT overexpression reduced podocyte damage, indicated by more podocytes, less glomerular cell death, and higher density of podocyte foot processes. In addition, expansion of glomerular and mesangial volume were significantly less in OVE26Nmt mice compared with OVE26 mice. Four-month-old OVE26Nmt mice had a 70 to 90% reduction in 24-h albumin excretion, but this protection does not seem to be permanent. These results provide evidence for the role of oxidative damage to the podocyte in diabetic mice and show that protection of the podocyte can reduce or delay primary features of diabetic nephropathy.

THERAPEUTIC EFFICACY OF SELEGILINE IN NEURODEGENERATIVE DISORDERS AND NEUROLOGICAL DISEASES

Selegiline inhibits the activity of monoamine oxidase B, enhances the release of dopamine, blocks the uptake of dopamine, acts as a calmodulin antagonist, and enhances the level of cyclic AMP, which in turn protects dopaminergic neurons. It possesses cognition-enhancing functions, rejuvenates serum insulin-like growth factor I in aged rats, and enhances life expectancy in rodents. Selegiline possesses neurotrophic-like actions, and rescues axotomized motorneurons independent of monoamine oxidase B inhibition. It enhances the synthesis of nerve growth factor, protects dopaminergic neurons from glutamate-mediated neurotoxicity, and protects dopaminergic neurons from toxic factors present in the spinal fluid of parkinsonian patients, and the said effect may be mediated via elaborating brain derived neurotrophic factor. Selegiline increases the striatal superoxide dismutase, protects against peroxynitrite- and nitric oxide-induced apoptosis, and guards dopaminergic neurons from toxicity induced by glutathione depletion. It stimulates the biosynthesis of interleukin 1-beta and interleukin-6, is an immunoenhancing substance, possesses antiapoptotic actions, and is neuroprotectant in nature. Selegiline has been shown to be efficacious in Parkinsons disease, global ischemia, Gille de la Tourette syndrome, and narcolepsy. Its therapeutic efficacy in Alzheimers disease remains uncertain. In Alzheimers disease, short term studies of selegiline suggest a beneficial effect; whereas long term studies are less convincing.

Podocyte Loss in Aging OVE26 Diabetic Mice

Recent studies show that podocyte nuclear density (NV) and numbers of renal podocytes per glomerulus (N) are altered in experimental and spontaneous diabetes mellitus. NV and N are generally reduced, and it has been hypothesized that these morphological changes may relate to the loss of glomerular permselectivity in diabetic nephropathy (DN). In the current study, OVE26 transgenic diabetic mice and age‐matched (FVB) controls (60, 150, or 450 days) were fixed by vascular perfusion and renal cortical tissues were prepared for morphometric analyses. ImageJ software and point counting analyses were carried out on light and transmission electron micrographs to determine glomerular volume (VG), NV, and N. As expected, mean VG in OVE26 mice increased substantially (∼134%) over the course of the study and was significantly increased over FVB mice at all ages. At 60 days, NV and N were not statistically distinguishable in OVE26 and control mice, while at 150 days, NV was significantly reduced in diabetics but not N. In 450‐day‐old OVE26 animals, however, NV and N were both significantly decreased (∼231% and ∼99%, respectively) relative to age‐matched FVB mice. These data suggest that in the OVE26 model of diabetes, significant podocyte loss occurs relatively late in the course of the disease. Moreover, it seems possible that these podocytic changes could play a role in sustaining the increased permeability of the blood–urine barrier in the later stages of diabetic renal decompensation. Anat Rec, 291:114–121, 2007.

Ultrastructural and functional analyses of nephropathy in calmodulin‐induced diabetic transgenic mice

Previous animal models of diabetic nephropathy have used diabetic animals for which the underlying defect was either uncertain or the diabetes was induced by potentially specific toxins. In this report, we describe the renal abnormalities in a transgenic mouse model that develops early‐onset diabetes due to overexpression of calmodulin in pancreatic beta cells.

1-Benzyl-1,2,3,4-tetrahydroisoquinoline, a Parkinsonism-inducing endogenous toxin, increases α-synuclein expression and causes nuclear damage in human dopaminergic cells

1‐Benzyl‐1,2,3,4‐tetrahydroisoquinoline (1BnTIQ), an endogenous neurotoxin, is known to cause parkinsonism in rodents and nonhuman primates. The levels of 1BnTIQ in cerebrospinal fluid of patients with Parkinsons disease (PD) were reported to be three times higher than those in control subjects. In the present study, we have evaluated the effects of 1BnTIQ on α‐synuclein (α‐syn) expression together with biochemical and morphological changes in human dopaminergic SH‐SY5Y cells in culture. 1BnTIQ at lower concentrations (1–50 μM) increased α‐syn protein expression in a time‐ and dose‐dependent manner in these cells. There was also up‐regulation of α‐syn mRNA by 1BnTIQ. Inhibition of complex I by rotenone and depletion of glutathione by L‐buthionine sulfoxamine also correlated with an increase in α‐syn expression, suggesting that oxidative stress may cause an increase in α‐syn levels in dopaminergic cells. Furthermore, 1BnTIQ significantly depleted glutathione levels. 1BnTIQ at higher concentrations (500 μM) increased reactive oxygen species levels, decreased ATP levels, and caused nuclear damage in the cells. The 1BnTIQ‐induced α‐syn up‐regulation was inhibited by cotreatment with the antioxidants selegiline, coenzyme Q10, and N‐acetylcystein and the caspase inhibitor DEVD‐CHO. Taken together, these results suggest that α‐syn up‐regulation and oxidative stress are contributing factors in 1BnTIQ‐induced neurotoxicity in dopaminergic neurons in PD.

Ultrastructure of intranuclear annulate lamellae in giant cells of rat placenta

The ultrastructure of intranuclear annulate lamellae (IAL) in placental giant cells of the rat was studied. Placentae, taken from Sprague—Dawlay rats on day 9 through day 14 of gestation, were fixed in 3 % glutaraldehyde, postfixed in 1 % osmic acid and embedded in Epon. IAL were rare in 9-day specimens and usually consisted of short lamellae closely associated with the internal nuclear envelope. They increased in frequency and size with time and became quite obvious in thick sections stained with toluidine blue. Electron micrographs revealed curved, linear, or branched profiles of IAL up to 8 μ in length. Continuity of their membranes with the internal nuclear membrane was occasionally evident. The pores of the IAL measured 1100–1600 A center-to-center and contained central dense granules. The IAL were surrounded by a moderately electron-opaque matrix in which granules 100–150 A in diameter could occasionally be observed. In one specimen IAL were in direct contact with a nucleolus. Here granules 140–160 A in diameter were arranged around the periphery of the IAL. These granules were separated from the IAL by a sleeve of matrix. The significance of these observations as related to the origin and function of IAL is discussed.

Electron microscopic studies of cat mesenteric arterioles: a structure-function analysis.

Abstract Arcade arterioles of cat mesentery were fixed in vivo at normal and elevated intravascular pressures and studied by transmission electron microscopy. In general, cells, filaments, and extracellular fibrils on the luminal side of the internal elastic lamina (IEL) were oriented longitudinally while those of the abluminal side were oriented circumferentially. The IEL presented a smooth, cylindrical appearance while the intima, media, and adventitia were quite irregular in cross section and longitudinal section. The vessel lumen contour was also irregular, especially in the vicinity of endothelial cell nuclei. Endothelial cell processes (ridges) projected through longitudinal slits (fenestrae) in the IEL and made contact with vascular smooth muscle (VSM) cells, often causing apparent deformation of the latter. VSM cells typically possessed multiple processes which were oriented circumferentially. Within the VSM cells myofibrils were oriented tangential to the vessel circumference. Dense bodies were preferentially located on the adventitial surface of the VSM but were also found at junctions between myocytes, suggesting a means of force transmission between cells. Vessels which were constricted with venous pressure elevation showed substantial changes in conformation of the wall structures, which may reflect considerable mobility of wall elements. Several areas of the VSM cell membrane appear to be deformed with intravascular pressure elevation, which may be important in the myogenic response to the pressure rise.

Immunoelectron microscopic analyses of Maillard reaction products in bovine anterior lens capsule and Descemet's membrane

It has been hypothesized that Maillard reaction products form in basement membranes during aging and may affect protein turnover. The purpose of this study was to localize Maillard reaction products in intact lens capsules and Descemets membranes by immunoelectron microscopy to determine whether Maillard products accumulated with age and whether basement membrane thickness increased to a similar degree. The monoclonal antibodies antiglucitollysine and antipyrraline were employed to detect the products in native and glucose-treated bovine basement membranes. The content of basic amino acids, furosine, and fluorophores (370/440), as well as resistance to trypsin digestion showed that the basement membranes formed significant quantities of Maillard products when incubated with 200 mM glucose in vitro (P < 0.05). Likewise, incubation in 200 mM glucose resulted in at least a 4-fold increase in immunoreactivity (P < 0.001). Native basement membranes increased in thickness more than 2-fold with age (P < 0.001). Immunoreactivity varied similarly in that bound antiglucitollysine increased approx. 2-fold and antipyrraline approx. 3-fold in old vs. young basement membranes, but these differences were significant only in pyrraline immunoreactivity in the lens capsule (P < 0.01). Advanced products other than pyrraline may accumulate in Descemets membrane since significant increases in fluorescence and resistance to trypsin were noted. These data suggest that the Maillard reaction may, to a small degree, contribute to basement membrane thickening.