Nutrients are key determinants of body size. In Drosophila
, nutrient-poor diets disrupt endoreplicative cell cycles, inhibit growth and delay metamorphosis (Britton and Edgar, 1998
; Colombani et al., 2003
). Fed dAMPKα
mutants live in a nutrient-rich environment, yet they bear similarities to starved animals. They are small, exhibit delayed pupariation and have low triglyceride levels, despite the role of AMPK as an activator of fatty acid oxidation and a negative regulator of lipogenesis through such targets as ACC. Furthermore, while loss of dAMPKα
mutant in the whole animal leads to reduced fat body cell size, loss of dAMPKα
in clones of fat body cells or in the whole eye has no effect on cell or organ size, indicating that dAMPK serves as a cell-nonautonomous regulator of growth. We find that dAMPKα
mutants are limited in their ability to store nutrients and grow because they fail to move food through their guts efficiently, leading to decreased nutrient absorption through the digestive tract.
Normal gut function requires contributions from polarized epithelial cells that absorb nutrients from food and muscle cells that grind and mix food and move it through the gut. In the mammalian intestine, maintenance of epithelial cell polarity contributes to the establishment of a protective barrier against the environment and to the digestion of nutrients via maintenance of electrochemical gradients for transport and appropriate localization of transporters. Maintenance of epithelial polarity is essential for gut function; flies lacking the gene Gp93
display altered gut epithelial polarity and exhibit a stunted-growth phenotype that resembles effects of starvation (Maynard et al., 2010
). Given the established role of dAMPK as a regulator of cell polarity (Lee et al., 2007
; Mirouse et al., 2007
), we examined the subcellular localization of proteins in normal and energetically-stressed gut epithelia. We find appropriate distribution of basolateral, lateral and apical proteins as well as an apical brush border throughout dAMPKα
mutant gut epithelia, indicating that AMPK is not required for maintenance of epithelial polarity in the gut, as is also the case in the Drosophila
eye (Amin et al., 2009
). We also observe an increased number of vacuoles in the dAMPKα
mutant midgut epithelia. This may reflect a compensatory increase in the digestive capacity of these cells. Indeed, microarray analysis shows increased expression of transcripts encoding digestive enzymes such as proteases, lipases and amylases in dAMPKα
mutant guts compared with wild type (MLB and MJB, unpublished data).
, AMPK acts in visceral muscle to promote peristalsis, thereby enhancing nutrient intake and supporting growth of the whole animal. Our data show that in Drosophila
, as in mammals, AMPK positively regulates muscle function (Hutber et al., 1997
; Minokoshi et al., 2002
; Mu et al., 2001
). Furthermore, by acting in visceral muscle to promote nutrient intake, dAMPK fulfills a physiological role that is mechanistically distinct from but functionally analogous to mammalian AMPK in the hypothalamus. In mice, activation of hypothalamic AMPK stimulates food intake (Kubota et al., 2007
; Minokoshi et al., 2004
). Our results demonstrate that AMPK performs an ancient, conserved role by acting as an important nonautonomous determinant of nutrient supply in flies and mammals.
Expression of wild type dAMPKα in muscle restores gut function and nutrient storage and permits survival through metamorphosis in dAMPKα
mutants. Surprisingly, muscle-rescued dAMPKα
mutants do not exhibit overgrowth phenotypes consistent with activation of the dTOR pathway, despite the negative regulation of mTOR by AMPK in mammalian cells (Gwinn et al., 2008
; Inoki et al., 2003
) and the dominant growth-promoting role played by dTOR in flies (reviewed in Edgar, 2006
). Therefore, while we cannot exclude that dAMPK inhibits dTOR during energy stress, at this point there is no evidence for a role of AMPK as an important basal regulator of TOR in vivo in flies. Our results indicate that regulation of visceral muscle function is the essential role of dAMPK during the larval stage of development.
Visceral muscle function and morphology are restored in whole-body dAMPKα
mutants by expression of a sqh transgene in which Thr21 and Ser22 are replaced by phosphorylation-mimicking glutamate residues. Expression of sqhEE
in visceral muscle may improve dAMPKα
mutant muscle morphology by promoting muscle contraction and thereby preventing atrophy or by correcting morphology and thereby restoring function; our data do not distinguish between these possibilities. An activated sqh transgene also rescues cell polarity defects in dAMPKα
-null embryos (Lee et al., 2007
), indicating that sqh acts downstream of or parallel to AMPK in multiple contexts in Drosophila
. In mammals, the sqh orthologue MRLC promotes smooth muscle contraction downstream of calcium signaling and myosin light chain kinase (MLCK) activation and G-protein coupled receptor signaling to Rho and Rok (Somlyo and Somlyo, 2003
). Additionally, AMPK regulates vascular smooth muscle contraction in mammals (Goirand et al., 2007
). The relationship between AMPK and MRLC in smooth muscle may be complex, though most data indicate that AMPK acts upstream of MRLC, either directly or indirectly through phosphorylation of MLCK (Bultot et al., 2009
; Horman et al., 2008
; Lee et al., 2007
). Our data are consistent with AMPK serving as a direct or indirect upstream regulator of MRLC or with AMPK acting in a pathway parallel to MRLC.
The signals that activate AMPK in visceral muscle remain obscure. We do not observe energy-dependent effects of food quality on gut function in Drosophila
larvae. Another possibility is that sustained nutrient absorption and visceral muscle contraction in the larval gut lead to high rates of ATP consumption, decreased energy charge and AMPK activation. Indeed, the metabolic activity of the digestive tract is estimated to account for 20% of whole-body oxygen consumption in mammals (Cant et al., 1996
). However, the rescue of dAMPKαΔ39
gut function with the activated MRLC transgene suggests that AMPK performs a regulatory role in visceral muscle and not that the gut fails in dAMPKα
mutants due to ATP depletion. Furthermore, whole-body lkb14B1–11
mutants do not exhibit the gut transit phenotype, despite low levels of dAMPKα phosphorylation. It may be that basal AMPK activity is sufficient to promote visceral muscle function in lkb14B1–11
mutants. Another possibility is that neuropeptides or neurotransmitters released by the stomatogastric nervous system might activate AMPK through kinases other than LKB1 and promote contraction (Nassel, 2002
). Many Drosophila
neuropeptides signal through Gq-coupled receptors, and this class of receptors activates AMPK in mammalian cells (Stahmann et al., 2006
; Zhang et al., 2008
AMPK mutant phenotype bears a striking resemblance to the human disease chronic idiopathic intestinal pseudo-obstruction (CIPO) (Stanghellini et al., 2007
). CIPO patients exhibit decreased intestinal mobility due to muscular or neural defects that impairs digestion, leading to severe malnutrition and, in many cases, lethality. Although mutations in genes encoding AMPK subunits have not been reported in CIPO patients, it will be of interest to determine whether AMPK or its upstream regulators might play a role in the progression of this disease and other visceral myopathies. If so, drugs that activate AMPK such as the commonly-prescribed, anti-diabetic metformin might be useful in treating these devastating conditions.
We have shown that in Drosophila, AMPK plays an essential role in regulating nutrient intake and supporting growth by acting in the visceral musculature to promote peristalsis. Our results identify a novel cell-nonautonomous function for AMPK in an invertebrate and underscore the importance of gut function in the regulation of organismal growth.