Fatty acid distribution in systems modeling the normal and diabetic human circulation. A 13C nuclear magnetic resonance study.

Cistola, David P.; Small, Donald

doi:10.1172/jci115149

Cited by 92 publications

(79 citation statements)

References 46 publications

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“…2A and Fig. S4A and B), with the strongest inhibition seen at a 6:1 palmitate-to-albumin ratio, a dose similar to what would be expected in obese or diabetic patients (30,31). Similar results were seen when GAPDH or tubulin were used as loading controls (not shown).…”

Section: Resultssupporting

confidence: 76%

Carboxypeptidase E mediates palmitate-induced β-cell ER stress and apoptosis

Jeffrey

Alejandro

Luciani

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

142

153

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Obesity is a principal risk factor for type 2 diabetes, and elevated fatty acids reduce ␤-cell function and survival. An unbiased proteomic screen was used to identify targets of palmitate in ␤-cell death. The most significantly altered protein in both human islets and MIN6 ␤-cells treated with palmitate was carboxypeptidase E (CPE). Palmitate reduced CPE protein levels within 2 h, preceding endoplasmic reticulum (ER) stress and cell death, by a mechanism involving CPE translocation to Golgi and lysosomal degradation. Palmitate metabolism and Ca 2؉ flux were also required for CPE proteolysis and ␤-cell death. Chronic palmitate exposure increased the ratio of proinsulin to insulin. CPE null islets had increased apoptosis in vivo and in vitro. Reducing CPE by Ϸ30% using shRNA also increased ER stress and apoptosis. Conversely, overexpression of CPE partially rescued ␤-cells from palmitate-induced ER stress and apoptosis. Thus, carboxypeptidase E degradation contributes to palmitate-induced ␤-cell ER stress and apoptosis. CPE is a major link between hyperlipidemia and ␤-cell death pathways in diabetes.2D difference gel electrophoresis proteomics ͉ free fatty acids ͉ hyperproinsulinemia ͉ mechanisms of ␤-cell lipotoxicity ͉ type 2 diabetes T here is a strong association between type 2 diabetes and obesity.High levels of circulating lipids, including free fatty acids, are a prominent clinical feature of type 2 diabetes and represent an important risk factor for this disease (1, 2). But exactly how elevated lipids might lead to diabetes remains unresolved. Fatty acids increase basal insulin secretion (3) and the relative levels of circulating proinsulin (4). Chronic exposure to the free fatty acid palmitate has been shown to impair glucose-stimulated insulin release (i.e., lipotoxicity) (5-10). ␤-Cell apoptosis can be initiated by high levels of palmitate (6,7,(11)(12)(13)(14), which may account in part for alterations in insulin secretory function (13). A number of studies have established palmitate targets in the ␤-cell, including lipid metabolism (15, 16), mitochondrial function (17-23), and prosurvival transcription factors such as Pdx1 (24,25). Recently, a role for endoplasmic reticulum (ER) stress in lipotoxicity has been demonstrated in multiple cell types, including ␤-cells (11,26,27). The effects of palmitate on ␤-cell survival are likely mediated by a number of mechanisms.In the present study, we conducted unbiased proteomic screens using human islets and MIN6 ␤-cells to elucidate targets of palmitate. Carboxypeptidase E (CPE) was the most significantly changed protein in both screens. Mice lacking CPE develop hyperproinsulinemia and hyperglycemia (28), but the involvement of this protein in ␤-cell apoptosis has not been reported. Palmitate caused the rapid intracellular redistribution and degradation of CPE via mechanisms that required palmitate metabolism, K ATP channel closure, Ca 2ϩ influx, and protease activity. We further showed that CPE levels control ␤-cell ER stress and apoptosis. Thus, CPE is a cri...

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Section: Resultssupporting

confidence: 76%

Carboxypeptidase E mediates palmitate-induced β-cell ER stress and apoptosis

Jeffrey

Alejandro

Luciani

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

142

153

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“…The progression of b-cell death events was simultaneously assessed using a live-cell assay that detected the temporal induction of plasma membrane blebbing, nuclear condensation, caspase-3 activation, annexin V incorporation, PI incorporation and loss of GFP. We characterized the kinetic interrelationships between cell death events in the context of multiple diabetes-associated conditions that can trigger the onset of b-cell death, including prolonged exposure to hyperglycemia, inflammatory cytokines and ER stress (Chu et al, 2010;Cistola and Small, 1991;Cnop et al, 2005;Federici et al, 2001;Maedler et al, 2001;Mathis et al, 2001). Although some kinetic features were common to all forms of b-cell death, we also found significant differences in event timing and order between distinct stresses.…”

Section: Discussionmentioning

confidence: 98%

“…Apoptosis is the main mode of cell death commonly assessed in the b-cell field (Chu et al, 2010;Cistola and Small, 1991;Cnop et al, 2005;Federici et al, 2001;Maedler et al, 2002;Maedler et al, 2001;Mathis et al, 2001), whereas contributions from other modes of cell death are often overlooked. Using our multiple-parameter single-cell analysis, we were able to distinguish between apoptotic, partial-apoptotic and non-apoptotic modes of primary b-cell death, which cannot be disassociated using cell population analyses of cell death (Fig.…”

Section: Primary B-cells Predominantly Undergo Non-apoptotic Cell Deathmentioning

confidence: 99%

Multi-parameter, single-cell, kinetic analysis reveals multiple modes of cell death in primary pancreatic beta-cells

Yang

Johnson

2013

Journal of Cell Science

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SummaryProgrammed b-cell death plays an important role in both type 1 and type 2 diabetes. Most of what is known about the mechanisms of bcell death comes from single time-point, single parameter measurements of bulk populations of mixed cells. Such approaches are inadequate for determining the true extent of the heterogeneity in death mechanisms. Here, we characterized the timing and order of molecular events associated with cell death in single b-cells under multiple diabetic stress conditions, including hyperglycemia, cytokine exposure, nutrient deprivation and endoplasmic reticulum (ER) stress. We simultaneously measured the kinetics of six distinct cell death mechanisms by using a caspase-3 sensor and three vital dyes, together with brightfield imaging. We identified several cell death modes where the order of events that usually define apoptosis were not observed. This we termed 'partial apoptosis'. Remarkably, complete classical apoptosis, defined as cells with plasma membrane blebbing, caspase-3 activity, nuclear condensation and membrane annexin V labeling prior to loss of plasma membrane integrity, was found in only half of the cytokine-treated primary b-cells and never in cells stressed by serum removal. By contrast, in the MIN6 cell line, death occurred almost exclusively through complete classical apoptosis. Ambient glucose modulated the cell death mode and kinetics in primary b-cells. Taken together, our data define the kinetic progression of b-cell death mechanisms under different conditions and illustrate the heterogeneity and plasticity of cell death modes in b-cells. We conclude that apoptosis is not the primary mode of adult primary b-cell death.

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“…The blood of diabetic humans can contain a ratio of up to 6:1 total fatty acids to albumin. Under such conditions, the total fatty acid can be Ͼ2000 M, where the unbound fatty acid can be Ͼ400 nM) (27). We chose a simplified in vitro approach where palmitate is the only fatty acid added to rapidly model chronic prediabetic or diabetic conditions.…”

Section: Methodsmentioning

confidence: 99%

ATP-Citrate Lyase Reduction Mediates Palmitate-induced Apoptosis in Pancreatic Beta Cells

Chu

Lin

Hendel

et al. 2010

Journal of Biological Chemistry

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Elevated extracellular lipids, such as the free fatty acid palmitate, can induce pancreatic beta cell endoplasmic reticulum (ER) stress and apoptosis, thereby contributing to the initiation and progression of type 2 diabetes. ATP-citrate lyase (ACLY), a key enzyme in cellular lipid production, was identified as a palmitate target in a proteomic screen. We investigated the effects of palmitate on ACLY activity and phosphorylation and its role in beta cell ER stress and apoptosis. We demonstrated that treatment of MIN6 cells, mouse islets and human islets with palmitate reduced ACLY protein levels. These in vitro results were validated by our finding that islets from high fat-fed mice had a significant decrease in ACLY, similar to that previously observed in type 2 diabetic human islets. Palmitate decreased intracellular acetyl-CoA levels to a similar degree as the ACLY inhibitor, SB-204990, suggesting a reduction in ACLY activity. ACLY inhibitors alone were sufficient to induce CCAAT/enhancer-binding protein homologues protein (CHOP)-dependent ER stress and caspase-3-dependent apoptosis. Similarly, even modest shRNA-mediated knockdown of ACLY caused a significant increase in beta cell apoptosis and ER stress. The effects of chemical ACLY inhibition and palmitate were nonadditive and therefore potentially mediated by a common mechanism. Indeed, overexpression of ACLY prevented palmitateinduced beta cell death. These observations provide new evidence that ACLY expression and activity can be suppressed by exogenous lipids and demonstrate a critical role for ACLY in pancreatic beta cell survival. These findings add to the emerging body of evidence linking beta cell metabolism with programmed cell death.

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Fatty acid distribution in systems modeling the normal and diabetic human circulation. A 13C nuclear magnetic resonance study.

Abstract: A nonperturbing '3C nuclear magnetic resonance (NMR) method was used to monitor the equilibrium distribution of carboxyl "C-enriched fatty acids (FA)

Cited by 92 publications

References 46 publications

Carboxypeptidase E mediates palmitate-induced β-cell ER stress and apoptosis

Carboxypeptidase E mediates palmitate-induced β-cell ER stress and apoptosis

Multi-parameter, single-cell, kinetic analysis reveals multiple modes of cell death in primary pancreatic beta-cells

ATP-Citrate Lyase Reduction Mediates Palmitate-induced Apoptosis in Pancreatic Beta Cells

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