digitonin and Obesity

The obesity-related 16 kDa peptide leptin is synthesized primarily in white adipocytes although its production has been reported in other tissues including the heart. There is emerging evidence that leptin may contribute to cardiac pathology especially that related to myocardial remodelling and heart failure. In view of the importance of mitochondria to these processes, the goal of the present study is to determine the effect of leptin on mitochondria permeability transition pore opening and the potential consequence in terms of development of apoptosis. Experiments were performed using neonatal rat ventricular myocytes exposed to 3.1 nM (50 ng/ml) leptin for 24 hours. Mitochondrial transition pore opening was analyzed as the capacity of mitochondria to retain the dye calcein-AM in presence of 200 µM CaCl2. Leptin significantly increased pore opening although the effect was markedly more pronounced in digitonin-permeabilized myocytes in the presence of calcium with both effects prevented by the transition pore inhibitor sanglifehrin A. These effects were associated with increased apoptosis as evidenced by increased TUNEL staining and caspase 3 activity, both of which were prevented by the transition pore inhibitor sanglifehrin A. Leptin enhanced Stat3 activation whereas a Stat 3 inhibitor peptide prevented leptin-induced mitochondrial transition pore opening as well as the hypertrophic and pro-apoptotic effects of the peptide. Inhibition of the RhoA/ROCK pathway prevented the hypertrophic response to leptin but had no effect on increased pore opening following leptin administration. We conclude that leptin can enhance calcium-mediated, Stat3-dependent pro-apoptotic effects as a result of increased mitochondrial transition pore opening and independently of its hypertrophic actions. Leptin may therefore contribute to mitochondrial dysfunction and the development of apoptosis in the diseased myocardium particularly under conditions of excessive intracellular calcium accumulation.

Topics: Animals; Apoptosis; Calcium; Digitonin; Hypertrophy; Leptin; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Myocytes, Cardiac; Obesity; Protein Conformation; Rats; Rats, Sprague-Dawley; rho-Associated Kinases; STAT3 Transcription Factor; Time Factors

Obesity-related diseases such as the metabolic syndrome and type 2 diabetes originate, in part, from the progressive metabolic deterioration of skeletal muscle. A preliminary proteomic survey of rectus abdominus muscle detected a statistically significant increase in adenylate kinase (AK)1, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and aldolase A in obese/overweight and morbidly obese women relative to lean control subjects. AK1 is essential for the maintenance of cellular energy charge, and GAPDH and aldolase A are well known glycolytic enzymes. We found that muscle AK1 protein and enzymatic activity increased 2.9 and 90%, respectively, in obese women and 9.25 and 100%, respectively, in morbidly obese women. The total enzymatic activity of creatine kinase, which also regulates energy metabolism in muscle, was shown to increase 30% in obese/overweight women only. We propose that increased protein and enzymatic activity of AK1 is representative of a compensatory glycolytic drift to counteract reduced muscle mitochondrial function with the progression of obesity. This hypothesis is supported by increased abundance of the glycolytic enzymes GAPDH and aldolase A in obese and morbidly obese muscle. In summary, proteome analysis of muscle has helped us better describe the molecular etiology of obesity-related disease.

Topics: Adenylate Kinase; Body Mass Index; Creatine Kinase; Digitonin; Electrophoresis, Gel, Two-Dimensional; Female; Humans; Middle Aged; Muscle Proteins; Obesity; Obesity, Morbid; Proteome; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Free Ca2+ was measured in intracellular stores of individual mouse pancreatic beta-cells using dual-wavelength microfluorometry and the low-affinity Ca2+ indicator furaptra. Controlled permeabilization of the plasma membrane with 4 micromol/l digitonin revealed that 22% of the furaptra was trapped in intracellular nonnuclear compartments. When 3 mmol/l ATP and 200 nmol/l Ca2+ were simultaneously present, this cation rapidly accumulated in the organelle pool, reaching an average concentration of 200-500 micromol/l. Whereas agents affecting the mitochondrial function (5 mmol/l succinate, 2 micromol/l ruthenium red, or 10 micromol/l antimycin A + 2 microg/ml oligomycin) had little effects, the Ca2+-ATPase inhibitor thapsigargin released 92% of the Ca2+ mobilizable with the ionophore Br-A23187. Digital imaging revealed regional differences in the organelle Ca2+. The regions with the highest Ca2+ concentration were particularly responsive to inositol 1,4,5-trisphosphate (IP3). IP3 mobilized Ca2+ in a dose-dependent way with half-maximal and maximal effects at about 1 and 5 micromol/l, respectively. High concentrations of IP3 released about half of the thapsigargin-sensitive Ca2+, but there were no responses to agents known to activate ryanodine receptors, such as 10 mmol/l caffeine, 0.1-1 micromol/l ryanodine, or 1-5 micromol/l cyclic ADP ribose. The results reinforce the concept that mobilization of intracellular Ca2+ in the pancreatic beta-cell is mediated by IP3 receptors rather than ryanodine receptors.

Topics: Animals; Calcimycin; Calcium; Cell Membrane; Cell Membrane Permeability; Digitonin; Enzyme Inhibitors; Fluorescent Dyes; Fura-2; Image Processing, Computer-Assisted; Inositol 1,4,5-Trisphosphate; Intracellular Membranes; Ionophores; Islets of Langerhans; Mice; Mice, Mutant Strains; Mitochondria; Obesity; Oligomycins