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Mitochondrial uncoupling protein-2 is not involved in palmitate-induced impairment of glucose-stimulated insulin secretion in INS-1E insulinoma cells and is not needed for the amplification of insulin release.

Hirschberg Jensen V, Affourtit C - Biochem Biophys Rep (2015)

Bottom Line: We demonstrate that there are no differences in temporal GSIS kinetics between perifused pseudoislets with and without UCP2.We conclude that UCP2 is not involved in palmitate-induced impairment of GSIS in INS-1E insulinoma cells and is not needed for the amplification of insulin release.These conclusions inform ongoing debate on the disputed biochemical and physiological functions of the beta cell UCP2.

View Article: PubMed Central - PubMed

Affiliation: School of Biomedical and Healthcare Sciences, Plymouth University, Drake Circus, Plymouth PL4 8AA, UK.

ABSTRACT

We have recently shown that overnight exposure of INS-1E insulinoma cells to palmitate in the presence of high glucose causes defects in both mitochondrial energy metabolism and glucose-stimulated insulin secretion (GSIS). Here we report experiments designed to test the involvement of mitochondrial uncoupling protein-2 (UCP2) in these glucolipotoxic effects. Measuring real-time oxygen consumption in siRNA-transfected INS-1E cells, we show that deleterious effects of palmitate on the glucose sensitivity of mitochondrial respiration and on the coupling efficiency of oxidative phosphorylation are independent of UCP2. Consistently, palmitate impairs GSIS to the same extent in cells with and without UCP2. Furthermore, we knocked down UCP2 in spheroid INS-1E cell clusters (pseudoislets) to test whether or not UCP2 regulates insulin secretion during prolonged glucose exposure. We demonstrate that there are no differences in temporal GSIS kinetics between perifused pseudoislets with and without UCP2. We conclude that UCP2 is not involved in palmitate-induced impairment of GSIS in INS-1E insulinoma cells and is not needed for the amplification of insulin release. These conclusions inform ongoing debate on the disputed biochemical and physiological functions of the beta cell UCP2.

No MeSH data available.


Related in: MedlinePlus

NEFA effects on absolute respiration in INS-1E cells±UCP2. Oxygen uptake was determined and normalised to cell number in INS-1E cells transfected with scrambled or Ucp2-targeted siRNA (white and grey bars, respectively). Before the respiratory assay, cells were incubated for 24 h in serum-deprived medium and exposed to BSA-conjugated NEFAs (PA=palmitate, POA=palmitoleate, PA+POA=palmitate and palmitoleate) or to BSA alone. During the respiratory assay, cells were incubated±28 mM glucose (panels B and A, respectively) and glucose-stimulated oxygen uptake was inhibited with 5 µg/mL oligomycin to distinguish respiration coupled to ATP synthesis from respiration associated with proton leak (panels C and D, respectively). Bars represent means±SEM of 4–5 separate respiratory assays and cell densities to normalise respiration were determined from DAPI-fluorescence (cf. [24]) in 3–4 parallel experiments. Each condition was probed 3–4 times in all experiments. Statistical significance of mean differences was tested by 2-way ANOVA with Sidak׳s multiple comparisons posthoc analysis: A differs from a (P<0.05 and P<0.0001 in panels B and C, respectively) and B differs from b (P<0.001).
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f0015: NEFA effects on absolute respiration in INS-1E cells±UCP2. Oxygen uptake was determined and normalised to cell number in INS-1E cells transfected with scrambled or Ucp2-targeted siRNA (white and grey bars, respectively). Before the respiratory assay, cells were incubated for 24 h in serum-deprived medium and exposed to BSA-conjugated NEFAs (PA=palmitate, POA=palmitoleate, PA+POA=palmitate and palmitoleate) or to BSA alone. During the respiratory assay, cells were incubated±28 mM glucose (panels B and A, respectively) and glucose-stimulated oxygen uptake was inhibited with 5 µg/mL oligomycin to distinguish respiration coupled to ATP synthesis from respiration associated with proton leak (panels C and D, respectively). Bars represent means±SEM of 4–5 separate respiratory assays and cell densities to normalise respiration were determined from DAPI-fluorescence (cf. [24]) in 3–4 parallel experiments. Each condition was probed 3–4 times in all experiments. Statistical significance of mean differences was tested by 2-way ANOVA with Sidak׳s multiple comparisons posthoc analysis: A differs from a (P<0.05 and P<0.0001 in panels B and C, respectively) and B differs from b (P<0.001).

Mentions: In line with our recent observations [17] overnight NEFA exposure at 11 mM glucose does not affect the absolute respiratory activity of INS-1E cells incubated without glucose (Fig. 3A). The lack of significant NEFA effect on this basal respiration is apparent in INS-1E cells that either contain or lack UCP2 (Fig. 3A). UCP2 depletion lowers the absolute basal respiratory rate in BSA control and palmitate-exposed cells, but these minor decreases are statistically insignificant (Fig. 3A). UCP2 knockdown does not affect absolute mitochondrial oxygen consumption stimulated by 28 mM glucose (Fig. 3B). Unlike non-transfected INS-1E cells [17], glucose-stimulated respiration is relatively insensitive to palmitate in cells transfected with scrambled siRNA (Fig. 3B) but, interestingly, UCP2 depletion augments palmitate sensitivity significantly (Fig. 3B). Palmitoleate lowers absolute glucose-stimulated respiration somewhat in scrambled-transfected cells, but this effect is only statistically significant when palmitoleate is added together with palmitate (Fig. 3B). In Ucp2-transfected cells, absolute glucose-stimulated respiration is insensitive to palmitoleate (Fig. 3B). Cells lacking UCP2 thus exhibit marginally higher glucose-stimulated mitochondrial respiratory rates after palmitoleate exposure (±palmitate) than cells with UCP2 (Fig. 3B).


Mitochondrial uncoupling protein-2 is not involved in palmitate-induced impairment of glucose-stimulated insulin secretion in INS-1E insulinoma cells and is not needed for the amplification of insulin release.

Hirschberg Jensen V, Affourtit C - Biochem Biophys Rep (2015)

NEFA effects on absolute respiration in INS-1E cells±UCP2. Oxygen uptake was determined and normalised to cell number in INS-1E cells transfected with scrambled or Ucp2-targeted siRNA (white and grey bars, respectively). Before the respiratory assay, cells were incubated for 24 h in serum-deprived medium and exposed to BSA-conjugated NEFAs (PA=palmitate, POA=palmitoleate, PA+POA=palmitate and palmitoleate) or to BSA alone. During the respiratory assay, cells were incubated±28 mM glucose (panels B and A, respectively) and glucose-stimulated oxygen uptake was inhibited with 5 µg/mL oligomycin to distinguish respiration coupled to ATP synthesis from respiration associated with proton leak (panels C and D, respectively). Bars represent means±SEM of 4–5 separate respiratory assays and cell densities to normalise respiration were determined from DAPI-fluorescence (cf. [24]) in 3–4 parallel experiments. Each condition was probed 3–4 times in all experiments. Statistical significance of mean differences was tested by 2-way ANOVA with Sidak׳s multiple comparisons posthoc analysis: A differs from a (P<0.05 and P<0.0001 in panels B and C, respectively) and B differs from b (P<0.001).
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f0015: NEFA effects on absolute respiration in INS-1E cells±UCP2. Oxygen uptake was determined and normalised to cell number in INS-1E cells transfected with scrambled or Ucp2-targeted siRNA (white and grey bars, respectively). Before the respiratory assay, cells were incubated for 24 h in serum-deprived medium and exposed to BSA-conjugated NEFAs (PA=palmitate, POA=palmitoleate, PA+POA=palmitate and palmitoleate) or to BSA alone. During the respiratory assay, cells were incubated±28 mM glucose (panels B and A, respectively) and glucose-stimulated oxygen uptake was inhibited with 5 µg/mL oligomycin to distinguish respiration coupled to ATP synthesis from respiration associated with proton leak (panels C and D, respectively). Bars represent means±SEM of 4–5 separate respiratory assays and cell densities to normalise respiration were determined from DAPI-fluorescence (cf. [24]) in 3–4 parallel experiments. Each condition was probed 3–4 times in all experiments. Statistical significance of mean differences was tested by 2-way ANOVA with Sidak׳s multiple comparisons posthoc analysis: A differs from a (P<0.05 and P<0.0001 in panels B and C, respectively) and B differs from b (P<0.001).
Mentions: In line with our recent observations [17] overnight NEFA exposure at 11 mM glucose does not affect the absolute respiratory activity of INS-1E cells incubated without glucose (Fig. 3A). The lack of significant NEFA effect on this basal respiration is apparent in INS-1E cells that either contain or lack UCP2 (Fig. 3A). UCP2 depletion lowers the absolute basal respiratory rate in BSA control and palmitate-exposed cells, but these minor decreases are statistically insignificant (Fig. 3A). UCP2 knockdown does not affect absolute mitochondrial oxygen consumption stimulated by 28 mM glucose (Fig. 3B). Unlike non-transfected INS-1E cells [17], glucose-stimulated respiration is relatively insensitive to palmitate in cells transfected with scrambled siRNA (Fig. 3B) but, interestingly, UCP2 depletion augments palmitate sensitivity significantly (Fig. 3B). Palmitoleate lowers absolute glucose-stimulated respiration somewhat in scrambled-transfected cells, but this effect is only statistically significant when palmitoleate is added together with palmitate (Fig. 3B). In Ucp2-transfected cells, absolute glucose-stimulated respiration is insensitive to palmitoleate (Fig. 3B). Cells lacking UCP2 thus exhibit marginally higher glucose-stimulated mitochondrial respiratory rates after palmitoleate exposure (±palmitate) than cells with UCP2 (Fig. 3B).

Bottom Line: We demonstrate that there are no differences in temporal GSIS kinetics between perifused pseudoislets with and without UCP2.We conclude that UCP2 is not involved in palmitate-induced impairment of GSIS in INS-1E insulinoma cells and is not needed for the amplification of insulin release.These conclusions inform ongoing debate on the disputed biochemical and physiological functions of the beta cell UCP2.

View Article: PubMed Central - PubMed

Affiliation: School of Biomedical and Healthcare Sciences, Plymouth University, Drake Circus, Plymouth PL4 8AA, UK.

ABSTRACT

We have recently shown that overnight exposure of INS-1E insulinoma cells to palmitate in the presence of high glucose causes defects in both mitochondrial energy metabolism and glucose-stimulated insulin secretion (GSIS). Here we report experiments designed to test the involvement of mitochondrial uncoupling protein-2 (UCP2) in these glucolipotoxic effects. Measuring real-time oxygen consumption in siRNA-transfected INS-1E cells, we show that deleterious effects of palmitate on the glucose sensitivity of mitochondrial respiration and on the coupling efficiency of oxidative phosphorylation are independent of UCP2. Consistently, palmitate impairs GSIS to the same extent in cells with and without UCP2. Furthermore, we knocked down UCP2 in spheroid INS-1E cell clusters (pseudoislets) to test whether or not UCP2 regulates insulin secretion during prolonged glucose exposure. We demonstrate that there are no differences in temporal GSIS kinetics between perifused pseudoislets with and without UCP2. We conclude that UCP2 is not involved in palmitate-induced impairment of GSIS in INS-1E insulinoma cells and is not needed for the amplification of insulin release. These conclusions inform ongoing debate on the disputed biochemical and physiological functions of the beta cell UCP2.

No MeSH data available.


Related in: MedlinePlus