News on CR, Glucose, Insulin

All:

One of the most distinct effects of CR is the lowering of integrated insulin and glucose levels, and it's widely believeed that one or the other or both play major roles in the anti-aging action of CR. To test this, one wants to be able to manipulate one orthe other or both, without lowering Caloric intake.

Famed CR and exercise researcher Roger McCarter has been doing some work with transgenic (TG) animals that overexpress the gene for GLUT4, the main glucose transporter in skeletal muscle. Basically, when insulin stimulates muscle, there is a cascade of molecular events which move GLUT4 to the cell surface, where they engulf glucose at the plasma and move it into the cell. By making more GLUT4, these mice suck up more glucose. Somewhat surprisingly, their insulin levels don't go down in homeostatic adjustment: they just have lower circulating glucose levels (and thus, presumably, fewer extracellular AGE).

In CR rodents and monkeys, "With brief CR, there is no change in insulin receptor number and binding affinity nor its tyrosine kinase activity, indicating a postinsulin receptor mechanism" for their improved insulin sensitivity (3). There does not seem to be any effect on the activity of the specific signal cascade components IRS-1, IRS-2, or phosphotyrosine-associated PI 3-kinase (3). GLUT4 LEVELS are unaltered, but translocation to the plasma membrane is increased (1,2); this may be related to increased phosphorylation (activation) of serine residues on Akt2 (3), ann insulin signaling component that lies downstream of PI 3-kinase.

One of the many exciting things on which you missed out if you didn't come out to the CR Society 2004 Conference was McCarter's old coinvestigator, Ed Masoro, informing us that McCarter has found that these GLUT4 transgenics have no changes in their lifespan. This suggests that, at a minimum, low glucose levels per se -- and extracellular AGE -- are not the *primary* mechanism of CR's anti-aging action, and may conceivably not contribute to it at all (but remember Karl Popper, and see discussion in (0). (Apparently, the bastard has been sitting on these results for ages, and just isn't interested in publishing his #$*()#!! results!).

Now, in results presented at the 2004 Gerontological Society of America meeting, they report that the lower glucose level does not appear to effect (OR affect!) CR-associated gene expression changes.

“we investigated altered gene expression by CR and the effect of low plasma glucose level, caused by the genetic manipulation rather than CR, on gene expression by measuring the level of mRNA expression in the liver tissues of young GLUT4 TG ad libitum fed (AL), GLUT4 TG CR, wild type littermates AL, and wild type littermates CR mice ... "

“The intersection of the results from three statistical analysis methods indicated 1,277 genes altered their expression by CR; however, none of them were altered in GLUT4 TG mice. In fact, the low plasma glucose level did not change much of any gene expression. Some of the array results were confirmed by real time quantitative reverse transcription polymerase chain reaction. We concluded that low plasma glucose level does not contribute to the beneficial effect of CR on gene expression.”

This was only an abstract, so there are a lot of details we'd want to know -- in particular, whether these are early- or long-term gene expression profiles. If the former, it would indeed suggest that low glucose does not play a role in any putative gene expression changes that are CAUSAL in the CR effect. If the latter, it would suggest that lower glucose does not prevent other events (enduring molecular lesions, eg) which lead to the age-related shifts in gene expression which CR PREVENTS by preventing prior events.

The distinction is very important and is obscured by most discussion on the subject.

I'd also like to see the methods they used to determine whether genes were or were not actually altered in expression by CR; previously-used methods have often been questionable, as in the early WEindruch/Prolla studies.

-Michael

0. de Grey AD.
Falsifying falsifications: the most critical task of theoreticians in biology.
Med Hypotheses. 2004;62(6):1012-20.
PMID: 15142666 [PubMed - in process]

1: Gazdag AC, Sullivan S, Kemnitz JW, Cartee GD.
Effect of long-term caloric restriction on GLUT4, phosphatidylinositol-3 kinase p85 subunit, and insulin receptor substrate-1 protein levels in rhesus monkey skeletal muscle.
J Gerontol A Biol Sci Med Sci. 2000 Jan;55(1):B44-6; discussion B47-8.
PMID: 10719762 [PubMed - indexed for MEDLINE]

2: Dean DJ, Brozinick JT Jr, Cushman SW, Cartee GD.
Calorie restriction increases cell surface GLUT-4 in insulin-stimulated
skeletal muscle.
Am J Physiol. 1998 Dec;275(6 Pt 1):E957-64.
PMID: 9843737 [PubMed - indexed for MEDLINE]

3. McCurdy CE, Davidson RT, Cartee GD.
Brief calorie restriction increases Akt2 phosphorylation in insulin-stimulated rat skeletal muscle.
Am J Physiol Endocrinol Metab. 2003 Oct;285(4):E693-700. Epub 2003 Jun 10.
PMID: 12799317 [PubMed - indexed for MEDLINE]

4. E. Han, M. Hickey, C. Fu, A. Richardson, L. Xi, R. Mccarter, S. Hilsenbeck, Y. Wu.
LOW PLASMA GLUCOSE LEVEL DOES NOT CONTRIBUTE TO THE BENEFICIAL EFFECT OF CALORIE RESTRICTION ON GENE EXPRESSION
Gerontologist. 2004 Oct; 44(1 Special):9-10.
http://www.eshow2000...racts_final.pdf

Good post. So when do we get to see all that good CR conference material online? Hint, hint...

Reason
Founder, Longevity Meme
reason@longevitymeme.org
http://www.longevitymeme.org

Michael, I have some questions:

1. Among the ~1,200 genes that CR altered, many of these are going to be the results of a "cascading effect", such that CR induces some primary gene expression alterations, and these in turn trigger the rest, right? What are some of the more important genes involved (e.g. SIR2) and would that be in this group of primary genes?
2. I am trying to understand this distinction between (i) GLUT4 failing to causally contribute to CR and (ii) GLUT4 failing to prevent things that CR prevents. I think the problem is that we are talking about counter-factuals. My best understanding is that we don't know whether, in this counter-factual world (which this research shows is not our world), GLUT would be a part of CR, or whether GLUT4 would be a partial-CR-surrogate, by preventing things before CR can. Is that correct?

This sounds like heresy:

Firstly, insulin is produced and secreted by pancreatic beta cells in response to rising glucose levels. Insulin then circulates via the bloodstream and binds to insulin receptors on muscle and adipocytes which signal the cell to activate GLUT4 glucose transporters to absorb glucose into the cell.

From the abstract of the paper

http://www.ncbi.nlm...._uids=15514260:

The 3 statistical analysis methods commonly indicated that FR altered the expression of 1277 genes; however, none of these genes was altered by additional GLUT4 expression. In fact, the low plasma glucose level in GLUT4 TG mice did not affect gene expression. Some results were confirmed by real-time quantitative RT-PCR. We conclude that a low plasma glucose level does not contribute to or coincide with the effect of FR on gene expression in the liver.

(FR = food restriction = CR)

Indeed having more GLUT4 glucose transporters will increase the rate of glucose absorption. By using GLUT4 transgenic (TG) mice whose skeletal muscle, cardiac muscle and adipose cells are genetically modified to enable them to produce more GLUT4 and therefore mop up glucose faster resulting in mice with a blood glucose level that is 20% lower than non TG mice. The theory of the investigators is that by keeping glucose levels down they are simulating the effects of CR. Ironically (?) they found that in these mice whose blood glucose levels were lower the effects of CR were not being induced.

Indeed having more GLUT4 glucose transporters will increase the rate of glucose absorption and lower the blood glucose levels but does not alter the insulin signaling from the pancreas and in fact increases glucose entry into the cell.

Having less glucose in the blood because it is being absorbed faster does not mean that there is less glucose entering the cell (in fact more glucose is entering the cell) and the insulin secretion following carbohydrate ingestion is not altered. Insulin is the ligand for DAF2 and glucose modulates SIR2.

Consequently, the interpretation that blood glucose levels regulate the beneficial CR mediated effects is wrong in the context of what is happening to the cell itself which is still exposed to the insulin signal and to intracellular glucose.

All:

Good post. So when do we get to see all that good CR conference material online? Hint, hint...

The plan is in fact to do this, although access may be restricted to full CR Society members. However, in the interim, Dean P's usually-accurate notes for Day 1, Day 2, and Day 3 are available.

CR Conferences really are a blast, esp if you're actually on CR: aside from the consistent high quality of the presentations, it's just delightful to hang around with people who get it.

-Michael