Autophagy in human type 2 diabetes pancreatic beta cells.
OBJECTIVE
Beta cell loss contributes to type 2 diabetes, with increased apoptosis representing an underlying mechanism. Autophagy, i.e. the physiological degradation of damaged organelles and proteins, may, if altered, be associated with a distinct form of cell death. We studied several features of autophagy in beta cells from type 2 diabetic patients and assessed the role of metabolic perturbation and pharmacological intervention.


METHODS
Pancreatic samples were obtained from organ donors and isolated islets prepared both by collagenase digestion and density gradient centrifugation. Beta cell morphology and morphometry were studied by electron microscopy. Gene expression studies were performed by quantitative RT-PCR.


RESULTS
Using electron microscopy, we observed more dead beta cells in diabetic (2.24 +/- 0.53%) than control (0.66 +/- 0.52%) samples (p < 0.01). Massive vacuole overload (suggesting altered autophagy) was associated with 1.18 +/- 0.54% dead beta cells in type 2 diabetic samples and with 0.36 +/- 0.26% in control samples (p < 0.05). Density volume of autophagic vacuoles and autophagosomes was significantly higher in diabetic beta cells. Unchanged gene expression of beclin-1 and ATG1 (also known as ULK1), and reduced transcription of LAMP2 and cathepsin B and D was observed in type 2 diabetic islets. Exposure of non-diabetic islets to increased NEFA concentration led to a marked increase of vacuole accumulation, together with enhanced beta cell death, which was associated with decreased LAMP2 expression. Metformin ameliorated autophagy alterations in diabetic beta cells and beta cells exposed to NEFA, a process associated with normalisation of LAMP2 expression.


CONCLUSIONS
Beta cells in human type 2 diabetes have signs of altered autophagy, which may contribute to loss of beta cell mass. To preserve beta cell mass in diabetic patients, it may be necessary to target multiple cell-death pathways.
Department of Experimental Pathology, University of Pisa, Pisa, Italy.
M
M
Masini
M
M
Bugliani
R
R
Lupi
S
S
del Guerra
U
U
Boggi
F
F
Filipponi
L
L
Marselli
P
P
Masiello
P
P
Marchetti
D000368:Aged; D051017:Apoptosis Regulatory Proteins / Q000235:genetics; D001343:Autophagy / Q000502:physiology*; D000071189:Autophagy-Related Protein-1 Homolog; D000071186:Beclin-1; D002401:Cathepsin B / Q000235:genetics; D002402:Cathepsin D / Q000235:genetics; D003924:Diabetes Mellitus, Type 2 / Q000188:drug therapy / Q000473:pathology*; D005260:Female; D006801:Humans; D050417:Insulin-Secreting Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology* / Q000648:ultrastructure*; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics; D052119:Lysosomal-Associated Membrane Protein 2; D051907:Lysosomal Membrane Proteins / Q000235:genetics; D008297:Male; D008565:Membrane Proteins / Q000235:genetics; D008687:Metformin / Q000494:pharmacology; D046529:Microscopy, Electron, Transmission; D008875:Middle Aged; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D051017:Apoptosis Regulatory Proteins; C491997:BECN1 protein, human; D000071186:Beclin-1; D047908:Intracellular Signaling Peptides and Proteins; C497811:LAMP2 protein, human; D052119:Lysosomal-Associated Membrane Protein 2; D051907:Lysosomal Membrane Proteins; D008565:Membrane Proteins; D008687:Metformin; D000071189:Autophagy-Related Protein-1 Homolog; D017346:Protein Serine-Threonine Kinases; C484754:ULK1 protein, human; D002401:Cathepsin B; D002402:Cathepsin D