Krishna M. Boini

Obesity: An overview on its current perspectives and treatment options

Obesity is a multi-factorial disorder, which is often associated with many other significant diseases such as diabetes, hypertension and other cardiovascular diseases, osteoarthritis and certain cancers. The management of obesity will therefore require a comprehensive range of strategies focussing on those with existing weight problems and also on those at high risk of developing obesity. Hence, prevention of obesity during childhood should be considered a priority, as there is a risk of persistence to adulthood. This article highlights various preventive aspects and treatment procedures of obesity with special emphasis on the latest research manifolds.

Mutation of the PDK1 PH Domain Inhibits Protein Kinase B/Akt, Leading to Small Size and Insulin Resistance

ABSTRACT PDK1 activates a group of kinases, including protein kinase B (PKB)/Akt, p70 ribosomal S6 kinase (S6K), and serum and glucocorticoid-induced protein kinase (SGK), that mediate many of the effects of insulin as well as other agonists. PDK1 interacts with phosphoinositides through a pleckstrin homology (PH) domain. To study the role of this interaction, we generated knock-in mice expressing a mutant of PDK1 incapable of binding phosphoinositides. The knock-in mice are significantly small, insulin resistant, and hyperinsulinemic. Activation of PKB is markedly reduced in knock-in mice as a result of lower phosphorylation of PKB at Thr308, the residue phosphorylated by PDK1. This results in the inhibition of the downstream mTOR complex 1 and S6K1 signaling pathways. In contrast, activation of SGK1 or p90 ribosomal S6 kinase or stimulation of S6K1 induced by feeding is unaffected by the PDK1 PH domain mutation. These observations establish the importance of the PDK1-phosphoinositide interaction in enabling PKB to be efficiently activated with an animal model. Our findings reveal how reduced activation of PKB isoforms impinges on downstream signaling pathways, causing diminution of size as well as insulin resistance.

Adaptogenic and nootropic activities of aqueous extract of Vitis vinifera (grape seed): an experimental study in rat model

BackgroundThe aerial parts of Vitis vinifera (common grape or European grape) have been widely used in Ayurveda to treat a variety of common and stress related disorders. In the present investigation, the seed extract of V. vinifera was evaluated for antistress activity in normal and stress induced rats. Furthermore, the extract was studied for nootropic activity in rats and in-vitro antioxidant potential to correlate its antistress activity.MethodsFor the evaluation of antistress activity, groups of rats (n = 6) were subjected to forced swim stress one hour after daily treatment of V. vinifera extract. Urinary vanillylmandelic acid (VMA) and ascorbic acid were selected as non-invasive biomarkers to assess the antistress activity. The 24 h urinary excretion of vanillylmandelic acid (VMA) and ascorbic acid were determined by spectrophotometric methods in all groups under normal and stressed conditions. The nootropic activity of the extract as determined from acquisition, retention and retrieval in rats was studied by conditioned avoidance response using Cooks pole climbing apparatus. The in vitro antioxidant activity was determined based on the ability of V. vinifera to scavenge hydroxyl radicals.ResultsDaily administration of V. vinifera at doses of 100, 200 and 300 mg/kg body weight one hour prior to induction of stress inhibited the stress induced urinary biochemical changes in a dose dependent manner. However, no change in the urinary excretion of VMA and ascorbic acid was observed in normal animals at all the doses studied. The cognition, as determined by the acquisition, retention and recovery in rats was observed to be dose dependent. The extract also produced significant inhibition of hydroxyl radicals in comparison to ascorbic acid in a dose dependent manner.ConclusionThe present study provides scientific support for the antistress (adaptogenic), antioxidant and nootropic activities of V. vinifera seed extract and substantiate the traditional claims for the usage of grape fruits and seeds in stress induced disorders.

Influence of NO Synthase Inhibitor L-NAME on Parasitemia and Survival of Plasmodium berghei Infected Mice

Accelerated suicidal death or eryptosis of infected erythrocytes may delay development of parasitemia in malaria. Eryptosis is inhibited by nitric oxide (NO). The present study has been performed to explore, whether inhibition of NO synthase by L-NAME modifies the course of malaria. We show here that L-NAME (>10 µM) increased phosphatidylserine exposure of Plasmodium falciparum infected human erythrocytes, an effect significantly more marked than in noninfected human erythrocytes. We further show that parasitemia in Plasmodium berghei infected mice was significantly decreased (from 50% to 18% of circulating erythrocytes 20 days after infection) by addition of 1 mg/ml L-NAME to the drinking water. According to CFSE labelling L-NAME treatment accelerated the clearance of both, noninfected and infected, erythrocytes from circulating blood, but did not significantly extend the life span of infected animals. In conclusion, treatment with L-NAME shortens the life span of circulating erythrocytes and thus delays development of parasitemia during malaria.

Influence of amitriptyline on eryptosis, parasitemia and survival of Plasmodium berghei-infected mice.

Chlorpromazine has previously been shown to trigger suicidal erythrocyte death or eryptosis, which is characterized by exposure of phosphatidylserine at the erythrocyte surface and cell shrinkage. Premature suicidal death of infected erythrocytes is in turn considered to delay development of parasitemia and thus favourably influence the clinical course of malaria. The present experiments have been performed to explore whether chlorpromazine influences in vitro parasite growth and eryptosis of Plasmodium falciparum infected human erythrocytes and in vivo parasitemia and survival of P. berghei infected mice. Phosphatidylserine was estimated from annexin V binding and cell volume from forward scatter in FACS analysis. In vitro infection of human erythrocytes increased annexin binding and decreased forward scatter, effects augmented in the presence of chlorpromazine (≧10 µM). Chlorpromazine did not significantly alter intraerythrocytic DNA/RNA content but significantly (≧1 µM) decreased in vitro parasitemia. In chlorpromazine treated mice erythrocytes were more rapidly cleared from circulating blood than in nontreated mice. Parasitemia in P. berghei infected mice was significantly decreased (from 50 % to 28 % of circulating erythrocytes 22 days after infection) and mouse survival significantly enhanced (from 0 % to 80 % 30 days after infection) by addition of 1 mM chlorpromazine to the drinking water from the first day of infection. In conclusion, chlorpromazine favourably influences the course of malaria, an effect at least partially due to stimulation of suicidal erythrocyte death.

Influence of Paclitaxel on Parasitemia and Survival of Plasmodium berghei Infected Mice

Paclitaxel triggers suicidal erythrocyte death or eryptosis, characterized by exposure of phosphatidylserine at the erythrocyte surface and cell shrinkage. Eryptosis of infected erythrocytes may delay development of parasitemia and thus favourably influence the course of malaria. The present study explored whether paclitaxel influences in vitro parasite growth and eryptosis of Plasmodium falciparum infected human erythrocytes and in vivo parasitemia and survival of Plasmodium berghei infected mice. Phosphatidylserine exposing erythrocytes were identified utilizing annexin V binding and erythrocyte volume was estimated from forward scatter in FACS analysis. In vitro infection of human erythrocytes with P. falciparum increased annexin binding and decreased forward scatter, effects augmented in the presence of paclitaxel (≥0.01 μM). Paclitaxel (≥0.01 μM) significantly decreased intraerythrocytic DNA/RNA content and in vitro parasitemia. In Plasmodium berghei infected mice parasitemia was significantly decreased (from 55.8% to 28.6% of circulating erythrocytes 20 days after infection) and mouse survival significantly enhanced (from 0% to 69.23% 25 days after infection) by administration of 8.5 mg/kg.b.w. of paclitaxel intraperitoneally from the eighth day of infection. In conclusion, paclitaxel decreases parasitemia and enhances survival of P. berghei infected mice, an effect, which may be due to stimulation of eryptosis and/or a direct toxic effect on the parasite.

Activation of Nod-Like Receptor Protein 3 Inflammasomes Turns on Podocyte Injury and Glomerular Sclerosis in Hyperhomocysteinemia

Inflammasome is a multiprotein complex consisting of Nod-like receptor protein 3 (NALP3), apoptosis-associated speck-like protein (ASC), and caspase 1 or 5, which functions to switch on the inflammatory process. The present study hypothesized that the formation and activation of NALP3 inflammasomes turn on podocyte injury leading to glomerulosclerosis during hyperhomocysteinemia (hHcys). RT-PCR and Western blot analysis demonstrated that murine podocytes expressed 3 essential components of the NALP3 inflammasome complex, namely, NALP3, ASC, and caspase 1. Treatment of podocytes with l-homocysteine induced the formation of NALP3 inflammasome complex, an increase in caspase 1 activity, podocyte cytoskeleton rearrangement, and decreased production of vascular endothelial growth factor from podocytes, which were all blocked by silencing the ASC gene or inhibiting caspase 1 activity. In mice with hHcys induced by feeding them a folate-free diet, NALP3 inflammasome formation and activation in glomerular podocytes were detected at an early stage, as shown by confocal microscopy, size exclusion chromatography of the assembled inflammasome complex, and increased interleukin-1&bgr; production in glomeruli. Locally silencing the ASC gene in the kidney significantly reduced NALP3 inflammasome formation and interleukin 1&bgr; production in glomeruli of mice with hHcys. Pathologically, hHcys-associated albuminuria, foot process effacement of podocytes, loss of podocyte slit diaphragm molecules, and glomerulosclerosis at the late stage were significantly improved by local ASC gene silencing or by caspase 1 inhibition. In conclusion, NALP3 inflammasome formation and activation on stimulation of homocysteine are important molecular mechanisms triggering podocyte injury and ultimately resulting in glomerulosclerosis in hHcys.

NADPH Oxidase-Mediated Triggering of Inflammasome Activation in Mouse Podocytes and Glomeruli During Hyperhomocysteinemia

AIM Our previous studies have shown that NOD-like receptor protein (NALP3) inflammasome activation is importantly involved in podocyte dysfunction and glomerular sclerosis induced by hyperhomocysteinemia (hHcys). The present study was designed to test whether nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-mediated redox signaling contributes to homocysteine (Hcys)-induced activation of NALP3 inflammasomes, an intracellular inflammatory machinery in podocytes in vitro and in vivo. RESULTS In vitro confocal microscopy and size-exclusion chromatography revealed that upon NADPH oxidase inhibition by gp91(phox) siRNA, gp91ds-tat peptide, diphenyleneiodonium, or apocynin, aggregation of inflammasome proteins NALP3, apoptosis-associated speck-like protein (ASC), and caspase-1 was significantly attenuated in mouse podocytes. This NADPH oxidase inhibition also resulted in diminished Hcys-induced inflammasome activation, evidenced by reduced caspase-1 activity and interleukin-1β production. Similar findings were observed in vivo where gp91(phox-/-) mice and mice receiving a gp91ds-tat treatment exhibited markedly reduced inflammasome formation and activation. Further, in vivo NADPH oxidase inhibition protected the glomeruli and podocytes from hHcys-induced injury as shown by attenuated proteinuria, albuminuria, and glomerular sclerotic changes. This might be attributed to the fact that gp91(phox-/-) and gp91ds-tat-treated mice had abolished infiltration of macrophages and T-cells into the glomeruli during hHcys. INNOVATION Our study for the first time links NADPH oxidase to the formation and activation of NALP3 inflammasomes in podocytes. CONCLUSION Hcys-induced NADPH oxidase activation is importantly involved in the switching on of NALP3 inflammasomes within podocytes, which leads to the downstream recruitment of immune cells, ultimately resulting in glomerular injury and sclerosis.

Role of Sphingolipid Mediator Ceramide in Obesity and Renal Injury in Mice Fed a High Fat Diet

The present study tested a hypothesis that excess accumulation of sphingolipid, ceramide, its metabolites, or a combination contributes to the development of obesity and associated kidney damage. Liquid chromatography/mass spectrometry analysis demonstrated that C57BL/6J mice on the high-fat diet (HFD) had significantly increased plasma total ceramide levels compared with animals fed a low-fat diet (LFD). Treatment of mice with the acid sphingomyelinase (ASMase) inhibitor amitriptyline significantly attenuated the HFD-induced plasma ceramide levels. Corresponding to increase in plasma ceramide, the HFD significantly increased the body weight gain, plasma leptin concentration, urinary total protein and albumin excretion, glomerular damage index, and adipose tissue ASMase activity compared with the LFD-fed mice. These HFD-induced changes were also significantly attenuated by treatment of mice with amitriptyline. In addition, the decline of plasma glucose concentration after an intraperitoneal injection of insulin (0.15 U/kg b.wt.) was more sustained in mice on the HFD with amitriptyline than on the HFD alone. Intraperitoneal injection of glucose (3 g/kg b.wt.) resulted in a slow increase followed by a rapid decrease in the plasma glucose concentration in LFD and HFD plus amitriptyline-treated mice, but such blood glucose response was not observed in HFD-fed mice. Immunofluorescence analysis demonstrated a decrease in the podocin and an increase in the desmin in the glomeruli of HFD-fed mice compared with the LFD and HFD plus amitriptyline-treated mice. In conclusion, our results reveal a pivotal role for ceramide biosynthesis in obesity, metabolic syndrome, and associated kidney damage.

Activation of Nlrp3 Inflammasomes Enhances Macrophage Lipid-Deposition and Migration: Implication of a Novel Role of Inflammasome in Atherogenesis

Although Nlrp3 inflammasome activation in macrophages has been shown to be critical for the development of atherosclerosis upon atherogenic stimuli, it remains unknown whether activated Nlrp3 inflammasomes by other non-atherogenic stimuli induce alterations in macrophages that may contribute in the concert with other factors to atherogenesis. Thus, the present study tested the hypothesis that activation of Nlrp3 inflammasomes by ATP, which is a classical non-lipid danger stimulus, enhances the migration of macrophage and increases lipids deposition in macrophages accelerating foam cell formation. We first demonstrated that extracellular ATP (2.5 mM) markedly increased the formation and activation of Nlrp3 inflammasomes in bone marrow macrophages (BMMs) from wild type (Asc+/+) mice resulting in activation of caspase-1 and IL-1β production. In these Asc+/+ macrophages, such stimulation of inflammasomes by non-lipid ATP was similar to those induced by atherogenic stimuli such as cholesterol crystals or 7-ketocholesterol. Both non-lipid and lipid forms of stimuli induced formation and activation of Nlrp3 inflammasomes, which were prevented by Asc gene deletion. Interestingly, Asc+/+ BMMs had dramatic lipids accumulation after stimulation with ATP. Further, we demonstrated that large amount of cholesterol was accumulated in lysosomes of Asc+/+ BMMs when inflammasomes were activated by ATP. Such intracellular and lysosomal lipids deposition was not observed in Asc−/− BMMs and also prevented by caspase-1 inhibitor WEHD. In addition, in vitro and in vivo experiments revealed that migration of Asc+/+ BMMs increased due to stimulation of Nlrp3 inflammasomes, which was markedly attenuated in Asc−/− BMMs. Together, these results suggest that activation of Nlrp3 inflammasomes remarkably increases the susceptibility of macrophages to lipid deposition and their migration ability. Such novel action of inflammasomes may facilitate entry or retention of macrophages into the arterial wall, where they form foam cells and ultimately induce atherosclerosis.