Key role for AMP-activated protein kinase in the ventromedial hypothalamus in regulating counterregulatory hormone responses to acute hypoglycemia.

OBJECTIVE: To examine in vivo in a rodent model the potential role of AMP-activated protein kinase (AMPK) within the ventromedial hypothalamus (VMH) in glucose sensing during hypoglycemia. RESEARCH DESIGN AND METHODS: Using gene silencing technology to selectively downregulate AMPK in the VMH, a key hypothalamic glucose-sensing region, we demonstrate a key role for AMPK in the detection of hypoglycemia. In vivo hyperinsulinemic-hypoglycemic (50 mg dl(-1)) clamp studies were performed in awake, chronically catheterized Sprague-Dawley rats that had been microinjected bilaterally to the VMH with an adeno-associated viral (AAV) vector expressing a short hairpin RNA for AMPKalpha. RESULTS: In comparison with control studies, VMH AMPK downregulation resulted in suppressed glucagon ( approximately 60%) and epinephrine (approximately 40%) responses to acute hypoglycemia. Rats with VMH AMPK downregulation also required more exogenous glucose to maintain the hypoglycemia plateau and showed significant reductions in endogenous glucose production and whole-body glucose uptake. CONCLUSIONS: We conclude that AMPK in the VMH plays a key role in the detection of acute hypoglycemia and initiation of the glucose counterregulatory response.

C. elegans microRNAs.

MicroRNAs (miRNAs) are small, non-coding regulatory RNAs found in many phyla that control such diverse events as development, metabolism, cell fate and cell death. They have also been implicated in human cancers. The C. elegans genome encodes hundreds of miRNAs, including the founding members of the miRNA family lin-4 and let-7. Despite the abundance of C. elegans miRNAs, few miRNA targets are known and little is known about the mechanism by which they function. However, C. elegans research continues to push the boundaries of discovery in this area. lin-4 and let-7 are the best understood miRNAs. They control the timing of adult cell fate determination in hypodermal cells by binding to partially complementary sites in the mRNA of key developmental regulators to repress protein expression. For example, lin-4 is predicted to bind to seven sites in the lin-14 3' untranslated region (UTR) to repress LIN-14, while let-7 is predicted to bind two let-7 complementary sites in the lin-41 3' UTR to down-regulate LIN-41. Two other miRNAs, lsy-6 and mir-273, control left-right asymmetry in neural development, and also target key developmental regulators for repression. Approximately one third of the C. elegans miRNAs are differentially expressed during development indicating a major role for miRNAs in C. elegans development. Given the remarkable conservation of developmental mechanism across phylogeny, many of the principles of miRNAs discovered in C. elegans are likely to be applicable to higher animals.

Architecture of a validated microRNA::target interaction.

MicroRNAs are small approximately 22 nucleotide regulators of numerous biological processes and bind target gene messenger RNAs to control gene expression. The C. elegans microRNA let-7 and its target lin-41 were the first microRNA::target interaction to be validated in vivo. let-7 molecules form imperfect duplexes with two required let-7 complementary sites in the lin-41 3' UTR. Here, we show that base pairing at both the 5' and 3' ends of the let-7 binding site, as well as the presence of unpaired RNA residues in the predicted duplexes, are required for lin-41 downregulation. In this study, our model for microRNA::target interactions also demonstrates that the context of a microRNA binding can be critical for function, revealing an unforeseen complexity in microRNA::target interactions.

The C. elegans microRNA let-7 binds to imperfect let-7 complementary sites from the lin-41 3'UTR.

Caenorhabditis elegans let-7, a founding member of the microRNA family, is predicted to bind to six sites in the 3'UTR of the mRNA of its target gene, lin-41, to down-regulate LIN-41. Here, we demonstrate that wild-type let-7 microRNA binds in vitro to RNA from the lin-41 3'UTR. This interaction is dependent on two conserved let-7 complementary sites (LCSs). A 27-nucleotide sequence between the LCSs is also necessary for down-regulation in vivo. LCS mutations compensatory to the lesion in let-7(n2853) can partially restore lin-41 3'UTR function in a let-7(n2853) background, providing the first experimental evidence for an animal miRNA binding directly to its validated target in vivo.

The C elegans hunchback homolog, hbl-1, controls temporal patterning and is a probable microRNA target.

hunchback regulates the temporal identity of neuroblasts in Drosophila. Here we show that hbl-1, the C. elegans hunchback ortholog, also controls temporal patterning. Furthermore, hbl-1 is a probable target of microRNA regulation through its 3'UTR. hbl-1 loss-of-function causes the precocious expression of adult seam cell fates. This phenotype is similar to loss-of-function of lin-41, a known target of the let-7 microRNA. Like lin-41 mutations, hbl-1 loss-of-function partially suppresses a let-7 mutation. The hbl-1 3'UTR is both necessary and sufficient to downregulate a reporter gene during development, and the let-7 and lin-4 microRNAs are both required for HBL-1/GFP downregulation. Multiple elements in the hbl-1 3'UTR show complementarity to regulatory microRNAs, suggesting that microRNAs directly control hbl-1. MicroRNAs may likewise function to regulate Drosophila hunchback during temporal patterning of the nervous system.