Chronic glucolipotoxic conditions in pancreatic islets impair insulin secretion due to dysregulated calcium dynamics, glucose responsiveness and mitochondrial activity
- Baggavalli P Somesh†1,
- Mahesh Kumar Verma†1,
- Manoj Kumar Sadasivuni†1,
- Anup Mammen-Oommen†1,
- Sanghamitra Biswas1,
- Pavagada C Shilpa1,
- Ashok Kumar Reddy1,
- Aggunda N Yateesh1,
- Puttrevana M Pallavi1,
- Siddaraju Nethra1,
- Rachapalli Smitha1,
- Korrapati Neelima1,
- Usha Narayanan1 and
- Madanahalli R Jagannath1Email author
© Somesh et al.; licensee BioMed Central Ltd. 2013
Received: 17 October 2012
Accepted: 24 June 2013
Published: 1 July 2013
In the progression towards diabetes, glucolipotoxicity is one of the main causes of pancreatic beta cell pathology. The aim of this study was to examine the in vitro effects of chronic glucolipotoxic conditions on cellular responses in pancreatic islets, including glucose and fat metabolism, Calcium mobilization, insulin secretion and insulin content.
Exposure of islets to chronic glucolipotoxic conditions decreased glucose stimulated insulin secretion in vitro. Reduced protein levels of Glut2/slc2a2, and decreased glucokinase and pyruvate carboxylase mRNA levels indicated a significant lowering in glucose sensing. Concomitantly, both fatty acid uptake and triglyceride accumulation increased significantly while fatty acid oxidation decreased. This general suppression in glucose metabolism correlated well with a decrease in mitochondrial number and activity, reduction in cellular ATP content and dampening of the TCA cycle. Further, we also observed a decrease in IP3 levels and lower Calcium mobilization in response to glucose. Importantly, chronic glucolipotoxic conditions in vitro decreased insulin gene expression, insulin content, insulin granule docking (to the plasma membrane) and insulin secretion.
Our results present an integrated view of the effects of chronic glucolipotoxic conditions on known and novel signaling events, in vitro, that results in reduced glucose responsiveness and insulin secretion.
KeywordsType 2 diabetes Rat islets Glucolipotoxicity Glucose metabolism Insulin content Insulin secretion
Type 2 diabetes mellitus (T2DM) is a metabolic disorder in which pancreatic insulin secretion does not meet the demands of insulin sensitivity [1, 2]. Over a period of time, consistently elevated levels of blood glucose and free fatty acids lead to glucolipotoxicity- mediated pancreatic beta cell dysfunction [3, 4]. It is now accepted that elevated glucose levels are required to mediate the lipotoxic effects, including inhibition of glucose-stimulated insulin secretion (GSIS), impaired insulin gene expression and apoptosis [4–8].
GSIS involves both glucose oxidation-coupled ATP production and the anaplerotic/cataplerotic pathway-mediated generation of coupling factors that trigger and amplify insulin secretion, respectively [9, 10]. Briefly, glucose uptake initiates metabolic pathways in which glucose is first converted to pyruvate mediated by glucokinase, and then to oxaloacetate by pyruvate carboxylase. Mitochondrial oxaloacetate generates citrate, a cataplerotic signal, which is transported to the cytosol and then broken down into acetyl-CoA initiating fatty acid synthesis. Acetyl-CoA is subsequently converted to malonyl-CoA, the concomitant step in fatty acid synthesis. In pancreatic beta cells, malonyl-CoA inhibits carnitine-palmitoyl transferase-1 (CPT-1) blocking fatty acid oxidation and resulting in the buildup of long-chain acyl-CoA esters (LC-CoA) in the cytosol . Long chain-CoA is thought to be a potential modulator of insulin secretion stimulating insulin granule docking and exocytosis [11, 12]. Glucose metabolism also raises the cytosolic ATP/ADP ratio, which inhibits the ATP-sensitive potassium channel (KATP) resulting in plasma membrane depolarization. In response to this, voltage-gated calcium channels open, causing an influx of extracellular calcium and exocytosis of insulin granules .
Another well-known role of glucose is augmenting insulin secretion by promoting phospholipase-C (PLC)-mediated hydrolysis of phosphatidylinositol 4, 5-biphosphate (PIP2) into diacylglycerol (DAG) and inositol triphosphate (IP3) . The DAG generated, in turn, activates protein kinase C (PKC), which is known to maintain insulin exocytosis [15, 16], while IP3 mobilizes calcium from endoplasmic reticulum stores. The PLC pathway is also known to upregulate cAMP levels in beta cells, which show glucose-mediated oscillations that correlate with insulin secretion [17, 18].
Further, glucose is known to increase insulin content through insulin gene transcription mediated by PDX1 and MAFa . Under normal conditions, the synthesized insulin is held in readily releasable pools which are transported to the plasma membrane by the small GTPase, Rab27a and the SNARE complex for acute calcium-mediated release [20, 21].
Chronic hyperglycemia (glucotoxicity) and hyperlipidemia (lipotoxicity) have been known to impair beta cell function [22, 23], and glucolipotoxicity has been defined as ‘the deleterious effects of elevated glucose and fatty acids on pancreatic beta cell-function and mass’ . Studies by Kashyap et al. in human subjects have shown that the ability of the beta cell to increase insulin secretion in response to fatty acids is a component that may predispose to T2DM . In accordance with this, animal models for T2DM show a glucolipotoxicity-mediated dysfunction in multiple cellular processes involved in insulin secretion [26–27 and references therein]. In vitro studies have been an important source of information to understand the molecular basis of glucolipotoxicity. For example, fatty acid-mediated inhibition of insulin gene transcription, which was identified in vitro, has been recapitulated in vivo. However, a known limitation of the in vitro studies in this area of research has been the varying concentrations of fatty acid used .
Here, we used specific concentrations of glucose and palmitate to study the effects of in vitro chronic glucolipotoxic conditions on intracellular signaling pathways and cellular processes that mediate glucose responsiveness and insulin secretion. We confirmed metabolic stress in pancreatic islets under these conditions using known stress markers. We found that chronic glucolipotoxicity impaired glucose and fat uptake/metabolism in rat pancreatic cells resulting in lower cellular ATP along with mitochondrial number and activity. In agreement with this, IP3 levels were also reduced as was the calcium mobilized by the IP3 receptor and the L-type voltage gated calcium channels. Finally, we found that chronic glucolipotoxicity significantly decreased insulin secretion by reducing both insulin gene expression and granule docking to the plasma membrane in pancreatic islets. Thus, our results present the first integrated view of glucolipotoxicity in vitro linking known and novel signaling events to reduced glucose sensitivity and insulin secretion.
To investigate the effects of chronic glucolipotoxicity on glucose responsiveness and insulin secretion, we generated glucolipotoxic conditions in rat pancreatic islets and the NIT1 beta cell line using 16.7 mM glucose and 500 μM palmitate.
Chronic glucolipotoxicity reduces insulin secretion in rat pancreatic islets
To understand the mechanism by which chronic glucolipotoxic conditions reduce GSIS in vitro, we next assessed glucose uptake/metabolism, calcium release, insulin gene expression and granule docking.
Glucose uptake and metabolism is impaired under chronic glucolipotoxic conditions
We next ascertained the link between malonyl-CoA formation and insulin secretion under chronic glucolipotoxic conditions. To this end, we treated rat islets cultured in glucolipotoxic conditions with high glucose and found a decrease in insulin secretion, as expected. Interestingly, when ATP citrate lyase (ACLY) was inhibited using radicicol , insulin secretion decreased further suggesting that ACLY and potentially the anaplerotic/cataplerotic pathways are involved in the dysregulation seen in insulin secretion (Figure 2F). Together, these results suggest that chronic glucolipotoxicity impairs glucose uptake and metabolism and thus, insulin secretion.
Chronic glucolipotoxicity impairs fatty acid uptake and metabolism
To ascertain whether fatty acid uptake is impaired under chronic glucolipotoxic conditions, we used a BODIPY dye, a non-metabolized fluorescently labelled fatty acid analog. We observed a three-fold increase in fatty acid uptake under chronic glucolipotoxic conditions (Figure 3C) indicating that along with CD36 mRNA and protein levels, fatty acid uptake was also impaired. Further, we also found fat metabolism to be impaired under chronic glucolipotoxic conditions as seen from the four-fold increase in triglyceride levels in the pancreatic beta cell line, NIT-1 (Figure 3D). This was validated by a reduction in fatty acid oxidation studied by measuring the mRNA levels of PPARa (Figure 3E). We confirmed that in vitro chronic glucolipotoxicity generated metabolic stress in the cell system using known markers of ER stress [28, 29] (Additional file 2: Figure S2A). Taken together, these data showed that chronic glucolipotoxic conditions impaired both glucose and fatty acid uptake and metabolism.
Mitochondrial number/activity and cytosolic ATP levels are reduced under chronic glucolipotoxic conditions
These data present the first line of evidence linking a decrease in cellular ATP to a reduction in mitochondrial number and activity under chronic glucolipotoxic conditions.
An increase in cytoplasmic calcium is required for insulin secretion under chronic glucolipotoxic conditions
As further confirmation, we ascertained whether L-type voltage gated calcium channels mobilized calcium under glucolipotoxic conditions by studying insulin secretion in the presence or absence of the L-type channel inhibitor, Nitrendipine, NTD . As reported earlier, we detected a decrease in high glucose-mediated secretion in the presence of NTD (Additional file 4: Figure S4A). In a similar assay, upon using the IP3 receptor inhibitor, 2-aminoethyldiphenyl borate (2-APB) , we found that endoplasmic reticulum calcium mobilization was also required for insulin secretion (Additional file 4: S4B). In summary, chronic glucolipotoxic conditions impaired IP3 levels and cytosolic calcium release.
Insulin synthesis and intracellular insulin content are reduced under chronic glucolipotoxic conditions
Insulin granule docking is reduced under chronic glucolipotoxic conditions
In animal models of T2DM, the small GTPase, Rab27a is known to be downregulated leading to decreased insulin granule docking to the plasma membrane, thereby lowering insulin secretion . In addition, insulin release at the fusion pore is also known to be impaired in diabetic animal models resulting in defects in exocytosis and release .
Taken together, these data provide the first, integrated in vitro view of known dysfunctional cellular mechanisms in chronic glucolipotoxic conditions, while identifying novel events such as the glucolipotoxicity-mediated reduction in mitochondrial number/activity and insulin granule docking/transport.
Despite intensive research, information about the mechanism of action and intracellular signaling pathways activated by glucolipotoxicity remains limited. Such an understanding has clinical relevance since the ability of the beta cell to increase insulin secretion in response to fatty acids is thought to be a predisposing factor for T2DM . In vitro studies have been important to gain a mechanistic understanding of glucolipotoxicity but have not allowed a complete view of glucolipotoxicity-mediated cellular dysregulation due to variations in the concentrations of fatty acids used . This study systematically evaluates specific in vitro glucolipotoxic conditions linking their effect to multiple cellular processes involved in insulin secretion and glucose responsiveness including glucose uptake/metabolism, fatty acid uptake/metabolism, cellular energetics, insulin synthesis, secretion and transport; and calcium dynamics.
We used 16.7 mM glucose and 500 μM palmitate in this study after evaluating multiple concentrations for their effect on metabolic stress and cell death (unpublished observations). Under these conditions, we confirmed metabolic stress in pancreatic islets and the NIT1 pancreatic beta cell line as seen by the strong induction of ER stress, oxidative stress and inflammation. As previously reported , these conditions also led to cell death as seen by the significant increase in caspase-3 activity (Additional file 2: Figure S2B).
Under chronic glucolipotoxic conditions in vitro, we found that insulin content and GSIS were lowered in rat pancreatic islets. Further, glucose and fat metabolism were impaired in islets correlating with the decrease in mitochondrial number/activity and cellular ATP levels. Chronic glucolipotoxicity reduced cytosolic calcium levels by decreasing calcium mobilization mediated by ITPR.
Our in vitro findings recapitulated data from previous glucolipotoxic studies in animal models showing an impact on glucose metabolism, calcium dynamics and insulin secretion/content [24, 44]. For example, we detected an increase in CD36 expression under glucolipotoxic conditions that correlated with enhanced triglyceride accumulation and reduced GSIS. These findings concur with data from cd36-null mouse models and over-expression studies in INS cell lines [45, 46] further validating the in vitro conditions used in our study.
We found it interesting that chronic glucolipotoxic conditions impacted multiple cellular processes including insulin synthesis, content and docking. On the basis of our results, we speculate that chronic glucolipotoxicity impacts insulin content most severely compared to insulin gene transcription, docking and secretion. Future studies will be required to get a more complete understanding of the same.
A key finding in our study is the influence of glucolipotoxicity on mitochondrial number/function. This is in line with the notion that enhanced insulin secretion may require an overall increase in mitochondrial activity/number as opposed to an isolated increase in an aspect of mitochondrial metabolism [47, 48]. In this study, we also detected a decrease in insulin granule docking/release under glucolipotoxic conditions indicative of a reduction in the readily releasable pool of insulin, which may have a bearing on the first-phase insulin secretion . Further studies are required to explore the mechanistic link between docking/exocytosis and the DAG-PKC pathway in more detail.
Increasing insulin secretion is an intensely pursued therapeutic strategy in T2DM. This study yields in vitro assay conditions that can be used to evaluate anti-diabetic agents, specifically insulin secretagogues, currently in development for their impact on glucolipotoxicity-mediated dysregulation. Importantly, an understanding of glucolipotoxicity-mediated cellular dysfunction may yield novel points of therapeutic intervention (i.e., targets/target classes) that hold promise in T2DM treatment. Thus, our study has potential to facilitate an improved understanding of pancreatic beta cell pathophysiology in T2DM.
Chronic glucolipotoxic conditions comprising high glucose and fatty acid resulted in various defects in key cellular machineries. Glucose sensing machinery involved in uptake and glucose metabolism for insulin secretion was reduced whereas fat uptake and triglyceride storage was increased. Defects in mitochondrial number and activity along with reduced ATP levels were observed under glucolipotoxic conditions. Similarly, beta cells showed increased ER stress, inflammation and apoptosis together with impaired calcium homeostasis. These defects occurred in conjunction with decreased insulin synthesis, insulin vesicle transport, docking and glucose-dependent insulin secretion. Our data provide a first integrated view of beta cell defects across multiple levels under chronic glucolipotoxic conditions.
RNA isolation, reverse transcription and quantitative real time polymerase chain reaction (qPCR)
Isolation and preparation of rat islets has been described in detail in Additional file 6 (supplementary methods). All animal studies and protocols were approved by the Institutional Animal Ethics Committee (IAEC) of Connexios Life Sciences Pvt Ltd. Post 72 h of incubation, total RNA was isolated and 1 μg of total RNA was used to generate cDNA (ABI, USA). Gene expression was measured using SYBR Green PCR Master Mix (Eurogenetic, Belgium). Gene primers for Slc2a2/Glut2, Gck, Pc, CD36, PPARα, Pdx1, Ins2, Rab27a, Il1β, Nos2a and Actb were based on mRNA sequences from the GenBank nucleotide database and designed in-house. Actb was used as an internal control. The primer sequence for the above gene markers are given in the Additional file 6 (supplementary methods).
Insulin secretion and content
Islets were isolated from rats (details in Additional file 6) and cultured in 90 mm petri-plates with RPMI 11 mM glucose and 10% FBS and penicillin streptomycin, in the presence or absence of 16.7 mM glucose and 500 μM palmitate for 72 h/37°C/5% CO2. Size-matched islets were isolated and transferred into 24-well plates containing 1ml KRBH (2.5 mM glucose)/well, and incubated at 37°C/5% CO2 for 1h. After removing the KRBH buffer, the islets were induced in KRBH buffer (250μl/well) at 37°C/5% CO2 for 2 h at indicated glucose concentrations with/without the specified pharmacological inhibitors. Inhibitors were used at the following concentrations: Radicicol (50 μM; Sigma) and Nitrendipine (5 μM; Sigma); 2-APB (10 μM, Sigma). Secreted insulin was measured in KRBH buffer using ELISA (Mercodia) as per manufacturer’s instructions. To measure insulin secretion in the presence of TCA cycle precursors, islets were prepared as above and treated with 5 mM leucine and 5 mM glutamine-containing KRBH for 2 h; 2 mM glucose without amino acids was used as a control. Islet lysates were used to measure intracellular insulin content and insulin levels were presented as ng insulin/islet.
NIT1 (ATCC) cells or rat islets were cultured with 5.5 mM glucose (control) with or without 16.7 mM glucose and 500 μM palmitate (GL) for 72 h. After incubation, cells or islets were lysed and total proteins were resolved by SDS-PAGE followed by transfer to nitrocellulose membrane. Protein expression and phosphorylation was measured using
Antibodies against Glut2, CD36 (Abcam), BiP, CHOP, p-eIF2a or β-actin (Cell Signaling Technology) and HRP conjugated secondary antibody (Bio-Rad). The protein specific signals were detected using chemiluminescence substrate (Pierce) and were quantified using Image-J software (NIH).
Measurement of glucose uptake
NIT1 cells were cultured with 5.5 mM glucose (control) with or without 16.7 mM glucose and 500 μM palmitate (GL) for 72 h. Post 72 h, cells were washed and incubated in glucose-free medium at 37°C for 30 min followed by incubation with 50 μM of 2-NBDG (2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose; Invitrogen) for 15min. After lysis, 2-NBDG uptake was measured at 465 nm excitation/540 nm emission, and normalized to total cellular DNA as measured using bis-benzamide at 360 nm.
Measurement of fatty acid uptake
NIT1 cells were cultured with 5.5 mM glucose (control) with or without 16.7 mM glucose and 500 μM palmitate (GL) for 72 h. Post 72 h, cells were washed and incubated in glucose-free medium at 37°C for 30 min followed by incubation with 1 μM of green-fluorescent BODIPY dyes (Molecular Probes) for 10 min. Cells were washed and incubated with 0.4% trypan blue for 5 min to quench any excess dye. Subsequently, cells were washed, lysed and BODIPY uptake was measured at 485 nm excitation/528 nm emission and normalized to total cellular proteins as measured using the Bradford assay (Bio-Rad). BODIPY uptake was represented as % of uptake under control condition.
Estimation of triglycerides
NIT1 cells were cultured with 5.5 mM glucose (control) with or without 16.7 mM glucose and 500 μM palmitate (GL) for 72 h. After incubation, cells were washed with PBS and lysed. Total cellular protein was estimated using the Bradford assay (Bio-Rad) and triglyceride levels were estimated using an enzymatic assay (DiaSys) as per manufacturer’s instructions. TAG levels were normalized to cellular protein levels.
Estimation of mitochondrial DNA copy number
Freshly isolated rat islets were cultured under control or glucolipotoxic conditions for 72 h. Post 72 h treatment, islets were harvested in digestion buffer (Tris-10mM, pH8.0, EDTA-1 mM, NaCl-5 mM, SDS-1% and RNAse-A-10 mg/ml) followed by a phenol-chloroform extraction and ethanol precipitation of total DNA. 5 μg DNA was used for quantitative real time PCR. Mitochondrial cytochrome C oxidase 1 (mtCox1) copy number was measured and normalized to nuclear DNA using hypoxanthine guanine phosphoribosyltransferase (HPRT).
Measurement of islet ATP
Rat islets were cultured as in the insulin secretion assay. Islets were incubated in the KRBH buffer containing 2.5 mM glucose for 1h followed by induction with 11 mM glucose (HG) for 1h. After 1 h of incubation, islets were lysed and ATP levels were estimated as per manufacturer’s instructions (ATP determination kit, Invitrogen).
Measurement of succinate dehydrogenase activity
NIT1 cells were cultured in 5.5 mM glucose (control) with or without 16.7 mM glucose and 500 μM palmitate (GL) for 72 h. After incubation, cells were washed and incubated in 100 mM potassium phosphate buffer containing 50 mM sucrose, 10 mM sodium azide, 500 mM sodium succinate and 8 mM INT (Iodonitrotetrazolium chloride; Sigma) for 2 h. Cells without sodium succinate were used as a negative control. After 2 h at 37°C, INT was dissolved in DMSO and estimated at 644 nm. The difference in absorbance with/ without succinate was calculated, normalized to total cellular protein and represented as % control SDH activity.
Estimation of islet IP3
Freshly isolated rat islets were cultured under normal condition (control) or under glucolipotoxic condition (GL) for 72 h. Islets were then washed and incubated in KRBH containing 2.5 mM glucose for 1h followed by treatment with HG for 5 min. IP3 levels were measured in the lysate using an immunoassay kit (Cusabio IP3 estimation kit).
Estimation of calcium mobilization
NIT1 cells were cultured with 5.5 mM glucose (control) with or without 16.7 mM glucose and 500 μM palmitate (glucolipotoxic) for 72 h. After incubation, cells were washed with calcium-free KRBH buffer followed by incubation at 37°C for 1 h in Fluo-3-AM calcium indicator fluorescent dye (Invitrogen). Cells were then induced with either low (2.5 mM) or high (16.7 mM) glucose and the fluorescence was measured at 485 nm. The baseline reading was established by reading fluorescence for 1 minute (from t=0 to t=1 minute) at 6 s intervals. The indicated glucose concentrations were added at t=1 minute. After mixing for 5 s, the final reading was taken for 3 minutes (from t=1 minute to t=4 min) at 6 s intervals.
Exocytosis of docked insulin granules
Islets were cultured as in the insulin secretion assay, washed and incubated in KRBH buffer containing 2.5 mM glucose for 1h. Islets were treated with low glucose alone (2 mM) with/without 30 mM KCl in KRBH for 30 min followed by estimation of secreted insulin in the buffer.
Data are expressed as mean±SEM and significance was calculated using the unpaired Student’s t-test. * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001 compared to the respective control. Unless mentioned otherwise, n=4 across experiments. Miscrosoft Excel was used for statistical analyses.
We would like to thank all the members of the in vitro experimentation group with specific mention of Bhawna Chandravanshi, Niketa Pawar, Aishwarya Sathyanarayan and Jayalaxmi Shetty at Connexios Life Sciences Pvt Ltd. for their assistance in the study. We would like to thank Dr.Lokesh Joshi, Dr. Raghavendra Rao, Dr. Mathiyazhagan and Aseem Premnath for their critical review of the manuscript and Dr. Rohit Kulkarni for helpful discussions.
These studies were supported by Connexios Life Sciences Pvt Ltd., a Nadathur Holdings company.
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