Publications by authors named "Neeta Khandekar"

10 Publications

  • Page 1 of 1

Use of a Hybrid Adeno-Associated Viral Vector Transposon System to Deliver the Insulin Gene to Diabetic NOD Mice.

Cells 2020 10 2;9(10). Epub 2020 Oct 2.

School of Life Sciences, University of Technology Sydney, 15 Broadway, Ultimo, NSW 2007, Australia.

Previously, we used a lentiviral vector to deliver furin-cleavable human insulin () to the livers in several animal models of diabetes using intervallic infusion in full flow occlusion (FFO), with resultant reversal of diabetes, restoration of glucose tolerance and pancreatic transdifferentiation (PT), due to the expression of beta (β)-cell transcription factors (β-TFs). The present study aimed to determine whether we could similarly reverse diabetes in the non-obese diabetic (NOD) mouse using an adeno-associated viral vector (AAV) to deliver - ± the β-TF to the livers of diabetic mice. The traditional AAV8, which provides episomal expression, and the hybrid AAV8/ that results in transgene integration were used. Diabetic mice that received AAV8- became hypoglycaemic with abnormal intraperitoneal glucose tolerance tests (IPGTTs). Expression of β-TFs was not detected in the livers. Reversal of diabetes was not achieved in mice that received AAV8--FUR and AAV8- and IPGTTs were abnormal. Normoglycaemia and glucose tolerance were achieved in mice that received AAV8/-/FFO. Definitive evidence of PT was not observed. This is the first in vivo study using the hybrid AAV8/ system to treat Type 1 diabetes (T1D). However, further development is required before the system can be used for gene therapy of T1D.
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http://dx.doi.org/10.3390/cells9102227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7600325PMC
October 2020

Cold exposure promotes obesity and impairs glucose homeostasis in mice subjected to a high‑fat diet.

Mol Med Rep 2018 Oct 10;18(4):3923-3931. Epub 2018 Aug 10.

Department of Cardiology, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing 400038, P.R. China.

Cold exposure is considered to be a form of stress and has various adverse effects on the body. The present study aimed to investigate the effects of chronic daily cold exposure on food intake, body weight, serum glucose levels and the central energy balance regulatory pathway in mice fed with a high‑fat diet (HFD). C57BL/6 mice were divided into two groups, which were fed with a standard chow or with a HFD. Half of the mice in each group were exposed to ice‑cold water for 1 h/day for 7 weeks, while the controls were exposed to room temperature. Chronic daily cold exposure significantly increased energy intake, body weight and serum glucose levels in HFD‑fed mice compared with the control group. In addition, 1 h after the final cold exposure, c‑fos immunoreactivity was significantly increased in the central amygdala of HFD‑fed mice compared with HFD‑fed mice without cold exposure, indicating neuronal activation in this brain region. Notably, 61% of these c‑fos neurons co‑expressed the neuropeptide Y (NPY), and the orexigenic peptide levels were significantly increased in the central amygdala of cold‑exposed mice compared with control mice. Notably, cold exposure significantly decreased the anorexigenic brain‑derived neurotropic factor (BDNF) messenger RNA (mRNA) levels in the ventromedial hypothalamic nucleus and increased growth hormone releasing hormone (GHRH) mRNA in the paraventricular nucleus. NPY‑ergic neurons in the central amygdala were activated by chronic cold exposure in mice on HFD via neuronal pathways to decrease BDNF and increase GHRH mRNA expression, possibly contributing to the development of obesity and impairment of glucose homeostasis.
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http://dx.doi.org/10.3892/mmr.2018.9382DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6131648PMC
October 2018

RANKL Reduces Body Weight and Food Intake via the Modulation of Hypothalamic NPY/CART Expression.

Int J Med Sci 2018 1;15(10):969-977. Epub 2018 Jul 1.

Department of Cardiology, Southwest Hospital, Third Military Medical University (Army Medical University), China.

The receptor activator of nuclear factorκB ligand (RANKL) modulates energy metabolism. However, how RANKL regulates energy homeostasis is still not clear. This study aims to investigate the central mechanisms by which central administration of RANKL inhibits food intake and causes weight loss in mice. We carried out a systematic and in-depth analysis of the neuronal pathways by which RANKL mediates catabolic effects. After intracerebroventricle () injection of RANKL, the expression of neuropeptide Y (NPY) mRNA in the Arc was significantly decreased, while the CART mRNA expression dramatically increased in the Arc and DMH. However, the agouti-related protein (AgRP) and pro-opiomelanocortin (POMC) mRNA had no significant changes compared with control groups. Together, the results suggest that central administration of RANKL reduces food intake and causes weight loss via modulating the hypothalamic NPY/CART pathways.
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http://dx.doi.org/10.7150/ijms.24373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6036154PMC
January 2019

Identification of liver-specific enhancer-promoter activity in the 3' untranslated region of the wild-type AAV2 genome.

Nat Genet 2017 Aug 19;49(8):1267-1273. Epub 2017 Jun 19.

Gene Therapy Research Unit, Children's Medical Research Institute and Sydney Children's Hospitals Network, University of Sydney, Sydney, New South Wales, Australia.

Vectors based on adeno-associated virus type 2 (AAV2) are powerful tools for gene transfer and genome editing applications. The level of interest in this system has recently surged in response to reports of therapeutic efficacy in human clinical trials, most notably for those in patients with hemophilia B (ref. 3). Understandably, a recent report drawing an association between AAV2 integration events and human hepatocellular carcinoma (HCC) has generated controversy about the causal or incidental nature of this association and the implications for AAV vector safety. Here we describe and functionally characterize a previously unknown liver-specific enhancer-promoter element in the wild-type AAV2 genome that is found between the stop codon of the cap gene, which encodes proteins that form the capsid, and the right-hand inverted terminal repeat. This 124-nt sequence is within the 163-nt common insertion region of the AAV genome, which has been implicated in the dysregulation of known HCC driver genes and thus offers added insight into the possible link between AAV integration events and the multifactorial pathogenesis of HCC.
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http://dx.doi.org/10.1038/ng.3893DOI Listing
August 2017

Reduced serum levels of oestradiol and brain derived neurotrophic factor in both diabetic women and HFD-feeding female mice.

Endocrine 2017 Apr 16;56(1):65-72. Epub 2016 Dec 16.

Department of Cardiology, Southwest Hospital, Third Military Medical University, Chongqing, China.

The estrogen levels in the pre and post menstrual phases interact with brain-derived neurotrophic factor in a complex manner, which influences the overall state of the body. To study the role of oestradiol and brain-derived neurotrophic factor in modulating obesity related type 2 diabetes and the interactions between two factors, we enrolled 15 diabetic premenopausal women and 15 diabetic postmenopausal women respectively, the same number of healthy pre and postmenopausal women were recruited as two control groups. The fasting blood glucose, insulin, lipids, estrogen, and brain-derived neurotrophic factor levels were measured through clinical tests. Additionally, we set up obese female mouse model to mimic human trial stated above, to verify the relationship between estrogen and brain-derived neurotrophic factor. Our findings revealed that there is a moderately positive correlation between brain-derived neurotrophic factor and oestradiol in females, and decreased brain-derived neurotrophic factor may worsen impaired insulin function. The results further confirmed that high fat diet-fed mice which exhibited impaired glucose tolerance, showed lower levels of oestradiol and decreased expression of brain-derived neurotrophic factor mRNA in the ventromedial hypothalamus. The level of brain-derived neurotrophic factor reduced on condition that the level of oestradiol is sufficiently low, such as women in postmenopausal period, which aggravates diabetes through feeding-related pathways. Increasing the level of brain-derived neurotrophic factor may help to alleviate the progression of the disease in postmenopausal women with diabetes.
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http://dx.doi.org/10.1007/s12020-016-1197-xDOI Listing
April 2017

Glycerol Monolaurate Inhibits Lipase Production by Clinical Ocular Isolates Without Affecting Bacterial Cell Viability.

Invest Ophthalmol Vis Sci 2016 Feb;57(2):544-50

Brien Holden Vision Institute Sydney, Australia 2School of Optometry and Vision Science, University of New South Wales, Sydney, Australia 3Vision Cooperative Research Centre, Sydney, Australia.

Purpose: We sought to determine the relative lipase production of a range of ocular bacterial isolates and to assess the efficacy of glycerol monolaurate (GML) in inhibiting this lipase production in high lipase-producing bacteria without affecting bacterial cell growth.

Methods: Staphylococcus aureus,Staphylococcus epidermidis,Propionibacterium acnes, and Corynebacterium spp. were inoculated at a density of 10(6)/mL in varying concentrations of GML up to 25 μg/mL for 24 hours at 37 °C with constant shaking. Bacterial suspensions were centrifuged, bacterial cell density was determined, and production of bacterial lipase was quantified using a commercial lipase assay kit.

Results: Staphylococcus spp. produced high levels of lipase activity compared with P. acnes and Corynebacterium spp. GML inhibited lipase production by Staphylococcal spp. in a dose-dependent manner, with S. epidermidis lipase production consistently more sensitive to GML than S. aureus. Glycerol monolaurate showed significant (P < 0.05) lipase inhibition above concentrations of 15 μg/mL in S. aureus and was not cytotoxic up to 25 μg/mL. For S. epidermidis, GML showed significant (P < 0.05) lipase inhibition above 7.5 μg/mL.

Conclusions: Lipase activity varied between species and between strains. Staphylococcal spp. produced higher lipase activity compared with P. acnes and Corynebacterium spp. Glycerol monolaurate inhibited lipase production by S. aureus and S. epidermidis at concentrations that did not adversely affect bacterial cell growth. GML can be used to inhibit ocular bacterial lipase production without proving detrimental to commensal bacteria viability.
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http://dx.doi.org/10.1167/iovs.15-17180DOI Listing
February 2016

The role of pancreatic polypeptide in the regulation of energy homeostasis.

Mol Cell Endocrinol 2015 Dec 27;418 Pt 1:33-41. Epub 2015 Jun 27.

Neurological Diseases Division, Research Program, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, Sydney, NSW 2010, Australia; School of Medical Sciences, University of New South Wales, Sydney, NSW 2052, Australia. Electronic address:

Imbalances in normal regulation of food intake can cause obesity and related disorders. Inadequate therapies for such disorders necessitate better understanding of mechanisms that regulate energy homeostasis. Pancreatic polypeptide (PP), a robust anorexigenic hormone, effectively modulates food intake and energy homeostasis, thus potentially aiding anti-obesity therapeutics. Intra-gastric and intra-intestinal infusion of nutrients stimulate PP secretion from the gastrointestinal tract, leading to vagal stimulation that mediates complex actions via the neuropeptide Y4 receptor in arcuate nucleus of the hypothalamus, subsequently activating key hypothalamic nuclei and dorsal vagal complex of the brainstem to influence energy homeostasis and body composition. Novel studies indicate affinity of PP for the relatively underexplored neuropeptide y6 receptor, mediating actions via the suprachiasmatic nucleus and pathways involving vasoactive intestinal polypeptide and insulin like growth factor 1. This review highlights detailed mechanisms by which PP mediates its actions on energy balance through various areas in the brain.
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http://dx.doi.org/10.1016/j.mce.2015.06.028DOI Listing
December 2015

Decrease in hyperosmotic stress-induced corneal epithelial cell apoptosis by L-carnitine.

Mol Vis 2013 19;19:1945-56. Epub 2013 Sep 19.

Brien Holden Vision Institute, Sydney, Australia.

Purpose: To characterize the osmoprotective properties of L-carnitine on human corneal epithelial cell volume and apoptosis during hyperosmotic stress.

Methods: Human corneal limbal epithelial (HCLE) cells were exposed to culture medium at 300 mOsm (isotonic) or 500 mOsm (hyperosmotic) with or without L-carnitine (10 mM). Induction of apoptosis was detected by quantifying the proteolytic activity of caspase-8, caspase-9, and caspase-3/7 using caspase activity assays, the expression of tumor necrosis factor (TNF)-α with enzyme-linked immunosorbent assay, and annexin V/propidium iodide staining of HCLE cells evaluated with confocal microscopy and flow cytometry. Cell volume changes in response to hyperosmotic stress were analyzed using flow cytometry.

Results: After the HCLE cells were exposed to hyperosmotic medium (500 mOsm), the percentage of shrunken cells and damaged/dead cells (stained positively for annexin V and/or propidium iodide) was six- and three-fold, respectively, higher than that under isotonic conditions (300 mOsm). This was paralleled by an increase in TNF-α concentration in media and caspase-8, -9, and -3/7 activities (six-, four-, ten-, and twelve-fold, respectively; all showing p < 0.001). Addition of L-carnitine during hyperosmotic stress partly restored cell volume and significantly reduced the concentration of TNF-α released (p = 0.005) and caspase-9 activity (p = 0.0125). Addition of L-carnitine reduced the percentage of hyperosmolarity-induced damaged/dead cells to levels observed under isotonic conditions.

Conclusions: L-carnitine can regulate human corneal epithelial cell volume under hyperosmotic stress and ameliorate hyperosmotic stress-induced apoptosis.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3782369PMC
March 2014

Betaine stabilizes cell volume and protects against apoptosis in human corneal epithelial cells under hyperosmotic stress.

Exp Eye Res 2013 Mar 12;108:33-41. Epub 2012 Dec 12.

Brien Holden Vision Institute, Sydney, Australia.

Elevated tear osmolarity is one of the key pathological factors in dry eye leading to ocular discomfort associated with damage to the ocular surface and inflammation. The aim of this study was to determine the capacity of the organic osmolyte, betaine, to act as an osmoprotectant against hypertonic stress-induced human corneal epithelial cell shrinkage and apoptosis using in vitro cell culture models. Human corneal limbal epithelial (HCLE) cells exposed to culture medium for 16 h at 300 mOsm (isotonic) or 500 mOsm (hyperosmotic) in the presence or absence of betaine (5 or 10 mM) were evaluated for cell volume changes; cell viability; and apoptosis. Betaine (10 mM) ameliorated hyperosmotically induced reduction of cell volume (from 27% reduction to 11%) and resulted in increased mitochondrial activity (by 17%) and an increase in viable cell numbers (by 12%) compared to controls (exposure to hyperosmotic medium without betaine). Hyperosmotically shocked HCLE cells in the presence of betaine (10 mM) halved the number of damaged cells (apoptotic/necrotic) compared to cells in the absence of betaine. The presence of betaine (at 5 or 10 mM) significantly reduced the activity of caspase-8, -9 and -3/7 and release of TNF-α was also reduced by 34% or 55% after exposure of HCLE to 500 mOsm in the presence of 5 or 10 mM betaine, respectively. Using polyclonal antibody against Betaine/GABA transporter 1 (BGT-1), we detected the presence of BGT-1 in HCLE. We demonstrated that the transport of betaine was facilitated by increased osmolarity. In conclusion, betaine stabilized corneal epithelial cell volume under hyperosmotic stress and limited hyperosmotic stress-induced HCLE apoptosis.
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http://dx.doi.org/10.1016/j.exer.2012.12.001DOI Listing
March 2013

Amyloid precursor proteins, neural differentiation of pluripotent stem cells and its relevance to Alzheimer's disease.

Stem Cells Dev 2012 May 18;21(7):997-1006. Epub 2012 Jan 18.

Faculty of Medicine, School of Psychiatry, University of New South Wales, Sydney, NSW, Australia.

Alzheimer's disease (AD) is a leading cause of age-related dementia that is characterized by an extensive loss of neurons and synaptic transmission. The pathological hallmarks of AD are neurofibrillary tangles and deposition of β-amyloid (Aβ) plaques. Previous research has investigated how Aβ fragments disrupt synaptic mechanisms in the vulnerable regions of the brain. There is a tremendous potential for stem cell technology to extend upon this research, not only in terms of developing therapeutic applications, but also in modeling AD. Indeed, the advent of induced pluripotent stem cell technology has opened up exciting new avenues for generating patient and disease-specific cell lines from somatic cells that may be used to model AD. Amyloid precursor protein (APP) is a key protein in neuronal development and this article reviews the role of APP in AD. Stem cell technology offers the opportunity to make use of APP in the directed differentiation of induced pluripotent stem cells into functional neurons, a process that may help generate a model of AD and thereby facilitate an understanding of the mechanisms underlying this disease.
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http://dx.doi.org/10.1089/scd.2011.0564DOI Listing
May 2012