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Sep 30, 2011

Role Identified for Cancer-Related Genetic Elements in Glucose Metabolism and Type 2 Diabetes

  • A role for genetic elements previously implicated in cancer progression has been found in the regulation of metabolism, insulin sensitivity, and the development of diabetes. The RNA-binding proteins Lin28a/b have been shown to promote malignancy by inhibiting let-7 biogenesis, but studies in experimental mice have now demonstrated that overexpression of Lin28a  or human LIN28B in mice promotes insulin sensitization that makes the animals resistant to developing high fat diet-induced diabetes.

    Conversely, the investigators claim, muscle-specific loss of Lin28a or overexpression of let-7 results in insulin resistance and impaired glucose tolerance. Led by George Q. Daley, M.D., at the Dana Faber Cancer Institute and Brigham and Women’s Hospital, the team says the changes in glucose tolerance and insulin sensitivity are controlled by let-7-mediated repression of the insulin-PI3K-mTOR pathway.

    Evaluation of GWAS further found that an enrichment of SNP-containing let-7 target genes is associated with type 2 diabetes and control of fasting glucose. Dr. Daley and colleagues report their findings in Cell in a paper titled “The Lin28/let-7 Axis Regulates Glucose Metabolism.”

    To investigate the in vivo function of the Lin28/let-7 pathway, the researchers generated transgenic mouse models. The team had previously generated a tetracycline-inducible Lin28a transgenic mouse (Lin28a Tg animals) that showed leaky constitutive Lin28a expression in the absence of induction.

    The young animals had been shown to exhibit enhanced glucose metabolism, so Dr. Daley's group investigated whether old Lin28a Tg mice might be resistant to age-induced obesity. Indeed, they found that animals in comparison with Lin28a Tg mice, wild-type mice fed a normal diet gained significantly more fat mass with age despite no evidence of differences in activity or food and water intake.

    When fed a high fat diet (HFD), the Lin28a Tg mice also consumed the same amount of food as wild-type animals but were more resistant to obesity, demonstrated markedly improved glucose tolerance and insulin sensitivity, and showed resistance to HFD-induced hepatosteatosis. These results provided the first indication that “Lin28a expression in the muscle, skin, and connective tissues protected against obesity and diabetes in the context of aging and HFD,” the authors write.

    Lin28a and Lin28b are differentially regulated even though they both block let-7 miRNAs, and in humans LIN28B is more frequently overexpressed in cancer than LIN28A. With this in mind the researchers generated a mouse strain carrying a doxycycline-inducible copy of human LIN28B (iLIN28B). Treatment of these animals to induce high levels of LIN28B production led to mature let-7 repression in organs, resulting in hypoglycaemia in dox-induced animals. Compared with control mice, the dox-induced iLIN28B mice also showed better glucose tolerance and insulin sensitivity when fed a normal diet, even though, they produced no more insulin than control littermates during glucose challenge.

    When fed a HFD, the iLIN28B mice demonstrated a marked and surprising resistance to weight gain, even though, they tended to eat more than the control animals, the researchers say. They add that the mice also continued to exhibit improved glucose tolerance. “These data show that both Lin28 homologs have similar effects on glucose metabolism and obesity, suggesting that these effects are mediated through common mRNA or miRNA targets of the Lin28 family,” the team states.

    To see if Lin28a is required physiologically for normal glucose metabolism in skeletal muscle, they then generated a skeletal muscle-specific Lin28a knockout. These tissue-specific knockouts demonstrated impaired glucose tolerance and insulin resistance, but there was no significant difference in let-7 levels during adult or embryonic stages. This suggested that Lin28a loss of function affects glucose homeostasis either through let-7-independent mRNA binding or through changes in the spatiotemporal distribution of let-7 miRNA.

    To test whether lack of let-7 expression had the opposite effect to  Lin28a/b gain of function, the researchers generated a mouse strain in which let-7g is doxycycline-inducible under the control of the Rosa26 locus. (iLet-7 mouse). These animals were in addition engineered so that let-7g was unable to bind to endogenous Lin-28.

    Global transgene induction from three weeks of age led to Let-7g overexpression, resulting in reduced body size and growth rates in induced animals. Let-7g-induced animals also demonstrated glucose intolerance whether the animals were fed a normal or HFD, and they produced more insulin than controls.

    “These results demonstrated that broad overexpression of let-7 results in peripheral glucose intolerance and compensatory overproduction of insulin from islet β cells,” the researchers remark.

    Interestingly, crossing the iLIN28B animals with the iLet-7 inducible mice and inducing LIN28B and let-7g had no effect on glucose tolerance, compared with either inducing LIN28B alone or let-7g alone. Thus, the combined findings suggested that LIN28 overexpression influences metabolism in part by suppressing let-7, whereas let-7 production alone is enough to regulate glucose metabolism in vivo.

    Studies in cell cultures showed that Lin28 expression results in a 50% increase in the uptake of glucose by myotubes, that this required activation of the PI3K-mTOR pathway, and that Lin28a-induction of Lin28a-induction of PI3K-mTOR signaling required exogenous growth factor stimulation. Interestingly, mTOR signaling could be suppressed by let-7 even in the absence of Lin28a , implying that  let-7 can act independently downstream of Lin28a, the authors note.

    The in vivo studies overall indicated that  “the effects of Lin28a on PI3K-mTOR signaling are at least in part due to let-7 and that Lin28 and let-7 exert opposing effects on PI3K-mTOR signaling.” This suggestion was further supported by the finding that the muscles of dox-induced iLIN28B mice showed increased levels of the targets of PI3K-mTOR signalling. The insulin-like growth factor 1 receptor (Igf1r) and the insulin receptor (Insr) proteins were also upregulated in muscles as a result of LIN28B induction, “reinforcing the fact that Lin28a/b drives insulin-PI3KmTOR signaling,” the team stresses.

    Because Lin28a was found to activate the insulin-PI3K-mTOR pathway both in vitro and in vivo, the researchers tested whether the metabolic effects of Lin28a in vivo could be inhibited by drugs that block the mTOR pathway. To this end they injected Lin28a Tg and wild-type littermates with rapamycin three times per week, starting when they were 18 days old.

    Rapamycin blocked the growth enhancement that the authors previous studies had shown occurred in Lin28a Tg mice, at doses that had little growth suppressive effects on wild-type animals. Short-term rapamycin treatment also reversed Lin28a-related enhancements to glucose uptake and reduced the insulin-sensitivity of Lin28a Tg mice to wild-type levels.

    When the team evaluated human genetic data to see whether the Lin28/let-7 pathway may be involved in type 2 diabetes, they found that numerous predicted let-7 target genes had been linked with the disease through genome-wide association studies and meta-analyses.

    “Of the computationally predicted let-7 targets associated with T2D, IGF2BP1/2/3 and Hmga2 have been verified as let-7 targets in several studies,” the authors remark. Looking more closely at these genes in cell culture experiments, they confirmed that cells increased the expression of Igf2bp1, Igf2bp2, and Hmga2 mRNA following Lin28a overexpression. Increased expression of Igf2bp2 and Igf2bp3 was also demonstrated in Lin28a Tg muscle, confirming the link in vivo.

    Further analysis of human genetic data suggested a more widespread connection between susceptibility to type 2 diabetes and let-7 targets in addition to those targets in validated type 2 diabetes association regions. Evaluation of data from the DIAGRAM+ meta-analysis of eight GWAS found significant enrichment of targets that includes genes overexpressed in response to let-7 overexpression.

    “The genes driving the type 2 diabetes enrichment signals for the different let-7 target sets include both functionally redundant homologs of T2D-associated genes such as IGF2BP1 (IGF2BP2), HMGA1 (HMGA2), DUSP12, and DUSP16 (DUSP9) and genes in the insulin-PI3K-mTOR pathway, including IRS2, INSR, AKT2, and TSC1,” Dr. Daley et al. note. Similarly, testing for enrichment of let-7 target gene associations with fasting glucose levels indicated an over-representation of multiple genes modestly associated with fasting glucose at different levels of signi?cance for the different let-7 target gene sets.

    “Our observation that the iLin28a, iLIN28B, and iLet-7 Tg gain of function mice as well as muscle-specific Lin28a loss of function mice manifest complementary phenotypes supports the notion that Lin28a/b and let-7 are both regulators of growth and developmental maturation,” the authors conclude. “PI3K/Akt signaling is known to promote Glut4 translocation to upregulate glucose uptake, while mTOR signaling can promote glucose uptake and glycolysis by changing gene expression independently of Glut4 translocation. Our report implicates Lin28a/b and let-7 as important modulators of glucose metabolism through interactions with the insulinPI3K-mTOR pathway and T2D-associated genes identified in GWAS.”

    The researchers admit that while their work implicates let-7 as a regulator of insulin-PI3K-mTOR signaling, it is also possible that there may be a role for direct mRNA targets of Lin28a/b in glucose metabolism.  Nevertheless, they stress, “although it is likely that additional mechanisms and feedback loops exist, our data suggests a model whereby Lin28a/b and let-7 coordinate the GWAS identified genes and the insulin-PI3K-mTOR pathway to regulate glucose. It also suggests that enhancing Lin28 function or abrogating let-7 may be therapeutically promising for diseases like obesity and diabetes.”


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