12 replies to this topic

[cudBwrong]

http://www.ncbi.nlm....pubmed/23874150

In the present study, resveratrol feeding had no effect on mitochondrial biogenesis in skeletal muscle even though our animals were fed a diet containing the same amount of resveratrol, 4 g/kg diet, as used by Lagouge et al. [3], and more than the dose, 0.4 g/kg diet, used by Bauer et al. [1]. In studies on the effects of resveratrol on cells in culture, concentrations in the 30 µM to 100 µM range have routinely been used [3],[12],[14],[15]. Based on our findings on C₂C₁₂ myotubes, the concentration of resveratrol required to induce an increase in mitochondrial biogenesis is above 10 µM, and the data shown by Bauer et al. [1] suggest the concentration of resveratrol needed to activate AMPK in CHO cells is also above 10 µM. The plasma resveratrol concentration in our rats was 1.56±0.28 µM and the highest concentration in the mice of Lagouge et al. [3] was ~0.5 µM. Thus, a likely explanation for the failure of resveratrol feeding to induce mitochondrial biogenesis in rats and mice in our study is its poor bioavailability.

In our experiments on C₂C₁₂ cells, we confirmed the finding of Lagouge et al. [3] that treatment of C₂C₁₂ cells with a high concentration of resveratrol results in both PGC-1α activation, evaluated using a PGC-1α–GAL4 construct together with a luciferase reporter, and an increase in mitochondrial biogenesis. The research groups of Auwerx and Puigserver have published a large number of studies, reporting that deacetylation activates and acetylation deactivates PGC-1α [3],[7],[15],[33]–[39]. Phosphorylation of PGC-1α by AMPK results in PGC-1 activation and increased mitochondrial biogenesis [22]. We found that high concentrations of resveratrol activate AMPK in C₂C₁₂ cells by a toxic effect on mitochondria that reduces ATP level, and that this is the mechanism by which resveratrol activates PGC-1α. We also found that the concomitant increase in SIRT1 activity, also mediated by AMPK, results in a deacetylation of PGC-1α that causes a blunting of the increase in PGC-1α activity induced by AMPK. This is in contrast to the report by Canto et al. [15] that activation of PGC-1α by AMPK is dependent on PGC-1α deacetylation by SIRT1. In support of this conclusion, they reported that inhibition of SIRT1 with nicotinamide or knock down of SIRT1 markedly reduced PGC-1α activation and attenuated the increase in mitochondrial proteins in response to AMPK activation.

[cudBwrong]

Curious and disturbing. A comment on pubmed, which is said to have been posted and "promptly removed" from PLoS Biology, points out inconsistencies in the data. The comment author claims it is not an allegation of misconduct, but it certainly raises troubling questions, especially as the data in question are Western Blots. Revisit the pubmed link, or read the quote below:

Fig. 1A, LCAD panel, lanes 2-4, appear identical to Fig. 1B LCAD panel lanes 1-3. Slightly rotated and with different exposure. This is despite these samples allegedly originating from different experimental treatments. Lots of shared features including the shapes and relative orientations of the bands, and the "kink" in the right most band, the relative shapes and orientations of the bands, and the "streak" emitting from the top right corner of the 3rd band.
Fig. 1A, PGC-1a panel (top one) appears identical to the PGC1a panel in Fig. 1B. They are simply different exposures of the same image, despite allegedly originating from different experimental treatments. Lots of shared features including the white spot in the lower part of the right-most band. The same appears true for the "SUO" blot (3rd from the top) - some "noise" spots added in panel A, but there's no doubt these are the same bands.
Fig. 4A (and elsewhere), some blots are used as loading controls for other blots, but cannot possibly have originated from the same gels, because some panels are spliced (as indicated by lines), and others are not. Sometimes it is permissible to run your samples on separate gels at the same time, in the exact same order, and then do the phospho vs. total blots separately and re-unite the data at the end. Here we are asked to believe the gels were run separately and with the samples in different order, then some of the blots were spliced (presumably to remove unwanted samples) but the others were not. This is not adherent to the usual standards of data presentation for this type of experiment.
Fig. 6B. The CYTO C panel appears to be simply a darker exposure of the one above it (COX IV). Lots of shared features, most notably the bubble above the right lane.
Fig. 3A, compare the band in the right lane of the cyto C panel (2nd blot from the top), with the band in the right lane of the CYTO C blot in Figure 4C. They are both of a shape that is too similar to be coincidental.
There are numerous other problems here.... the entire paper contains 85 (!!!) panels of western blotting data, every single one of which is "letter-boxed" to show only the band of interest, and none of which are annotated with molecular weight markers. In addition, despite the common origin of most of the samples, there is variability in the properties of the bands. For example in Fig. 4A, the phospho-ACC blot has 2 bands but the total ACC blot only has one band. Why? In Fig. 5B the ATPase blot has 3 bands, but only a single band in Fig. 3B. In several other cases, enhancing the contrast of the western blots reveals that adjacent bands have completely different color histograms - some are grayscale while others are color. As such, it is difficult to believe that these bands originated from the same scanned blot images (which is a prerequisite for being able to splice together blots).
Note... these are NOT allegations of any type of misconduct. I'm just pointing out what appears to be a very sloppy attitude toward data presentation, which seems to have resulted in a number of the "wrong" western blot images ending up in the published paper. Hey, with 85 almost identical looking images to keep track of, there were bound to be a few that slipped through the net! I'm sure these can easily be attributed to mistakes during electronic figure preparation, and corrected in a manner that in no way affects the conclusions of the paper.

[etizsupplyusa.com]

Is it mitochondrial DNA Resveratrol is supposed to effect the expression of?

[cudBwrong]

Is it mitochondrial DNA Resveratrol is supposed to effect the expression of?

I think this is one of the effects being considered. There are many reports, sometimes conflicting, on the effects of resveratrol on mitochondria. Since DNA expression, in general, is the process by which gene information is used to create a gene product, the many studies concerning differences in mitochondria behavior in the presence of resveratrol have something to do with gene expression.

Here is a recent one, picked almost at random:

http://www.ncbi.nlm....pubmed/24178296

Here, the authors find that resveratrol causes mitochondria to behave differently, in vitro and in vivo. There is more "mitochondrial complex I activity." But there seem to be multiple mechanisms which can be influenced by many different factors. Our knowledge of this is still emerging.

J Biol Chem. 2013 Dec 20;288(51):36662-75. doi: 10.1074/jbc.M113.466490. Epub 2013 Oct 31.
Resveratrol Induces a Mitochondrial Complex I-dependent Increase in NADH Oxidation Responsible for Sirtuin Activation in Liver Cells.

Desquiret-Dumas V, Gueguen N, Leman G, Baron S, Nivet-Antoine V, Chupin S, Chevrollier A, Vessières E, Ayer A, Ferré M, Bonneau D, Henrion D, Reynier P, Procaccio V.

Abstract


Resveratrol (RSV) has been shown to be involved in the regulation of energetic metabolism, generating increasing interest in therapeutic use. SIRT1 has been described as the main target of RSV. However, recent reports have challenged the hypothesis of its direct activation by RSV, and the signaling pathways remain elusive. Here, the effects of RSV on mitochondrial metabolism are detailed both in vivo and in vitro using murine and cellular models and isolated enzymes. We demonstrate that low RSV doses (1-5 μm) directly stimulate NADH dehydrogenases and, more specifically, mitochondrial complex I activity (EC50 ∼1 μm). In HepG2 cells, this complex I activation increases the mitochondrial NAD(+)/NADH ratio. This higher NAD(+) level initiates a SIRT3-dependent increase in the mitochondrial substrate supply pathways (i.e. the tricarboxylic acid cycle and fatty acid oxidation). This effect is also seen in liver mitochondria of RSV-fed animals (50 mg/kg/day). We conclude that the increase in NADH oxidation by complex I is a crucial event for SIRT3 activation by RSV. Our results open up new perspectives in the understanding of the RSV signaling pathway and highlight the critical importance of RSV doses used for future clinical trials.

[etizsupplyusa.com]

Is it mitochondrial DNA Resveratrol is supposed to effect the expression of?

I think this is one of the effects being considered. There are many reports, sometimes conflicting, on the effects of resveratrol on mitochondria. Since DNA expression, in general, is the process by which gene information is used to create a gene product, the many studies concerning differences in mitochondria behavior in the presence of resveratrol have something to do with gene expression.

Here is a recent one, picked almost at random:

http://www.ncbi.nlm....pubmed/24178296

Here, the authors find that resveratrol causes mitochondria to behave differently, in vitro and in vivo. There is more "mitochondrial complex I activity." But there seem to be multiple mechanisms which can be influenced by many different factors. Our knowledge of this is still emerging.

J Biol Chem. 2013 Dec 20;288(51):36662-75. doi: 10.1074/jbc.M113.466490. Epub 2013 Oct 31.
Resveratrol Induces a Mitochondrial Complex I-dependent Increase in NADH Oxidation Responsible for Sirtuin Activation in Liver Cells.

Desquiret-Dumas V, Gueguen N, Leman G, Baron S, Nivet-Antoine V, Chupin S, Chevrollier A, Vessières E, Ayer A, Ferré M, Bonneau D, Henrion D, Reynier P, Procaccio V.

Abstract


Resveratrol (RSV) has been shown to be involved in the regulation of energetic metabolism, generating increasing interest in therapeutic use. SIRT1 has been described as the main target of RSV. However, recent reports have challenged the hypothesis of its direct activation by RSV, and the signaling pathways remain elusive. Here, the effects of RSV on mitochondrial metabolism are detailed both in vivo and in vitro using murine and cellular models and isolated enzymes. We demonstrate that low RSV doses (1-5 μm) directly stimulate NADH dehydrogenases and, more specifically, mitochondrial complex I activity (EC50 ∼1 μm). In HepG2 cells, this complex I activation increases the mitochondrial NAD(+)/NADH ratio. This higher NAD(+) level initiates a SIRT3-dependent increase in the mitochondrial substrate supply pathways (i.e. the tricarboxylic acid cycle and fatty acid oxidation). This effect is also seen in liver mitochondria of RSV-fed animals (50 mg/kg/day). We conclude that the increase in NADH oxidation by complex I is a crucial event for SIRT3 activation by RSV. Our results open up new perspectives in the understanding of the RSV signaling pathway and highlight the critical importance of RSV doses used for future clinical trials.

Thank you for giving me some background and some links to go off of! It's appreciated my friend

Evan

[hav]

Looks like this study involved both rats and mice. Although it's primarily concerned with resveratrol effects on muscle, it didn't mention logging anything other than feeding presumably sedentary subjects a high fat diet plus resveratrol for 8 weeks and then sacrificing them to chemically analyze their muscles. Guess they proved resveratrol isn't a very good exercise mimic for couch potatoes.

Howard

Edited by hav, 08 January 2014 - 07:56 PM.

[cudBwrong]

Looks like this study involved both rats and mice. Although it's primarily concerned with resveratrol effects on muscle, it didn't mention logging anything other than feeding presumably sedentary subjects a high fat diet plus resveratrol for 8 weeks and then sacrificing them to chemically analyze their muscles. Guess they proved resveratrol isn't a very good exercise mimic for couch potatoes.

Howard

Howard, what do you think about the questions raised in the comment about the data integrity? Blow up the figures and take a look. I think the LCAD might be a problem, maybe not, but the PGC-1 alpha definitely looks problematic to me. Figures 1A and 1B look like the same image, but I think they are supposed to be different experiments.

[hav]

Howard, what do you think about the questions raised in the comment about the data integrity? Blow up the figures and take a look. I think the LCAD might be a problem, maybe not, but the PGC-1 alpha definitely looks problematic to me. Figures 1A and 1B look like the same image, but I think they are supposed to be different experiments.

Didn't really know what to make of that. Got the impression that the critic thought it was just sloppy editing. As opposed to a Dr Das thing. But I guess a sloppy paper assembly might reflect on overall lab and data management.

Don't understand the subjective process of interpreting a Western Blot myself... looks suspiciously like reading tea leaves to me. Even assuming their bar charts and numeric data are objectively correct, I still prefer seeing studies measuring more tangible things. In this case they may have gotten different results if they allowed their subjects to exercise, perhaps recording some measure of their performance like mileage, before doing their muscle composition analysis.

Howard

Edited by hav, 08 January 2014 - 10:50 PM.

[bixbyte]

One. Anyone ever purchase Resveratrol from India?
Two. Purity of the Resveratol?
Three. Chart of the plasma levels?

[maxwatt]

Yes. IF it's from Orchid Pharmaceutical, they did at the time I bought some, use methanol as a solvent, and they synthesized it chemically from precursors. Prohibitively expensive process, and if I recall the taste was a bit off. Some of the solvent was still present, though a low amount at 99% purity.

If your potential source is making it by extraction from knotweed, watch out for lead contamination. Every sample I know about from that Indian knotweed does not meet ISO standards for lead, and exceeded the standard by quite a bit.

Ii it is being produced by bacterial fermentation, it is probably OK, but it depends on the source's facility.

If they claim to be producing it from grapes or grape skin, and the price is competitive, then there might be some grape product in it, but most of it is probably a cheap synthetic from bacteria.

[Wilmore Labs]

This is why we now have pubpeer: 

 

https://pubpeer.com/...9FD58693CA41560

 

The story continues. 

 

Curious and disturbing. A comment on pubmed, which is said to have been posted and "promptly removed" from PLoS Biology, points out inconsistencies in the data. The comment author claims it is not an allegation of misconduct, but it certainly raises troubling questions, especially as the data in question are Western Blots. Revisit the pubmed link, or read the quote below:
 

 

Fig. 1A, LCAD panel, lanes 2-4, appear identical to Fig. 1B LCAD panel lanes 1-3. Slightly rotated and with different exposure. This is despite these samples allegedly originating from different experimental treatments. Lots of shared features including the shapes and relative orientations of the bands, and the "kink" in the right most band, the relative shapes and orientations of the bands, and the "streak" emitting from the top right corner of the 3rd band.
Fig. 1A, PGC-1a panel (top one) appears identical to the PGC1a panel in Fig. 1B. They are simply different exposures of the same image, despite allegedly originating from different experimental treatments. Lots of shared features including the white spot in the lower part of the right-most band. The same appears true for the "SUO" blot (3rd from the top) - some "noise" spots added in panel A, but there's no doubt these are the same bands.
Fig. 4A (and elsewhere), some blots are used as loading controls for other blots, but cannot possibly have originated from the same gels, because some panels are spliced (as indicated by lines), and others are not. Sometimes it is permissible to run your samples on separate gels at the same time, in the exact same order, and then do the phospho vs. total blots separately and re-unite the data at the end. Here we are asked to believe the gels were run separately and with the samples in different order, then some of the blots were spliced (presumably to remove unwanted samples) but the others were not. This is not adherent to the usual standards of data presentation for this type of experiment.
Fig. 6B. The CYTO C panel appears to be simply a darker exposure of the one above it (COX IV). Lots of shared features, most notably the bubble above the right lane.
Fig. 3A, compare the band in the right lane of the cyto C panel (2nd blot from the top), with the band in the right lane of the CYTO C blot in Figure 4C. They are both of a shape that is too similar to be coincidental.
There are numerous other problems here.... the entire paper contains 85 (!!!) panels of western blotting data, every single one of which is "letter-boxed" to show only the band of interest, and none of which are annotated with molecular weight markers. In addition, despite the common origin of most of the samples, there is variability in the properties of the bands. For example in Fig. 4A, the phospho-ACC blot has 2 bands but the total ACC blot only has one band. Why? In Fig. 5B the ATPase blot has 3 bands, but only a single band in Fig. 3B. In several other cases, enhancing the contrast of the western blots reveals that adjacent bands have completely different color histograms - some are grayscale while others are color. As such, it is difficult to believe that these bands originated from the same scanned blot images (which is a prerequisite for being able to splice together blots).
Note... these are NOT allegations of any type of misconduct. I'm just pointing out what appears to be a very sloppy attitude toward data presentation, which seems to have resulted in a number of the "wrong" western blot images ending up in the published paper. Hey, with 85 almost identical looking images to keep track of, there were bound to be a few that slipped through the net! I'm sure these can easily be attributed to mistakes during electronic figure preparation, and corrected in a manner that in no way affects the conclusions of the paper.

 

 

 

[amark]

I have seen different in early studies.

[maxwatt]

Depends on the dose, likely there is a threshold.