LKB1 is required for hepatic bile acid transport and canalicular membrane integrity in mice

LKB1 is a ‘master’ protein kinase implicated in the regulation of metabolism, cell proliferation, cell polarity and tumorigenesis. However, the long-term role of LKB1 in hepatic function is unknown. In the present study, it is shown that hepatic LKB1 plays a key role in liver cellular architecture and metabolism. We report that liver-specific deletion of LKB1 in mice leads to defective canaliculi and bile duct formation, causing impaired bile acid clearance and subsequent accumulation of bile acids in serum and liver. Concomitant with this, it was found that the majority of BSEP (bile salt export pump) was retained in intracellular pools rather than localized to the canalicular membrane in hepatocytes from LLKB1KO (liver-specific Lkb1-knockout) mice. Together, these changes resulted in toxic accumulation of bile salts, reduced liver function and failure to thrive. Additionally, circulating LDL (low-density lipoprotein)-cholesterol and non-esterified cholesterol levels were increased in LLKB1KO mice with an associated alteration in red blood cell morphology and development of hyperbilirubinaemia. These results indicate that LKB1 plays a critical role in bile acid homoeostasis and that lack of LKB1 in the liver results in cholestasis. These findings indicate a novel key role for LKB1 in the development of hepatic morphology and membrane targeting of canalicular proteins

Integrated genomic approaches implicate osteoglycin (Ogn) in the regulation of left ventricular mass

Left ventricular mass (LVM) and cardiac gene expression are complex traits regulated by factors both intrinsic and extrinsic to the heart. To dissect the major determinants of LVM, we combined expression quantitative trait locus1 and quantitative trait transcript (QTT) analyses of the cardiac transcriptome in the rat. Using these methods and in vitro functional assays, we identified osteoglycin (Ogn) as a major candidate regulator of rat LVM, with increased Ogn protein expression associated with elevated LVM. We also applied genome-wide QTT analysis to the human heart and observed that, out of 22,000 transcripts, OGN transcript abundance had the highest correlation with LVM. We further confirmed a role for Ogn in the in vivo regulation of LVM in Ogn knockout mice. Taken together, these data implicate Ogn as a key regulator of LVM in rats, mice and humans, and suggest that Ogn modifies the hypertrophic response to extrinsic factors such as hypertension and aortic stenosi

LKB1 is an essential regulator of spermatozoa release during spermiation in the mammalian testis

LKB1 acts as a master upstream protein kinase regulating a number of kinases involved in diverse cellular functions. Recent studies have suggested a role for LKB1 in male fertility. Male mice with reduced total LKB1 expression, including the complete absence of the major splice variant in testis (LKB1(S)), are completely infertile. We sought to further characterise these mice and determine the mechanism underlying this infertility. This involved expression studies of LKB1 in developing germ cells, morphological analysis of mature spermatozoa and histological studies of both the testis and epididymis using light microscopy and transmission electron microscopy. We conclude that a defect in the release of mature spermatids from the seminiferous epithelium (spermiation) during spermatozoan development is a major cause of the infertility phenotype. We also present evidence that this is due, at least in part, to defects in the breakdown of the junctions, known as ectoplasmic specialisations, between the sertoli cells of the testis epithelium and the heads of the maturing spermatids. Overall this study uncovers a critical role for LKB1 in spermiation, a highly regulated, but poorly understood process vital for male fertility

AMP-activated protein kinase (AMPK) is a tau kinase, activated in response to amyloid β-peptide exposure

International audienceHyperphosphorylation of tau is a hallmark of Alzheimer's disease and other tauopathies. Although the mechanisms underlying hyperphosphorylation are not fully understood, cellular stresses such as impaired energy metabolism are thought to influence the signalling cascade. The AMPK-related kinases, MARK and BRSK, have been implicated in tau phosphorylation but are insensitive to activation by cellular stress. Here we show that AMPK itself phosphorylates tau on a number of sites including S262 and S396, altering microtubule binding of tau. In primary mouse cortical neurons, CaMKKβ activation of AMPK in response to β-amyloid (Aβ1-42) leads to increased phosphorylation of tau at S262/S356 and S396. Activation of AMPK by Aβ1-42 is inhibited by memantine, a partial antagonist of the N-methyl-D-aspartate (NMDA) receptor and currently licensed for the treatment of Alzheimer's disease. These findings identify a pathway in which Aβ1-42 activates CaMKKβ and AMPK via the NMDA receptor suggesting the possibility that AMPK plays a role in the pathophysiological phosphorylation of tau

Histology of the cauda and caput epididymis.

<p>Cauda epididymis (<b>A</b>–<b>B</b>) and caput epididymis (<b>C</b>–<b>D</b>) sections were visualised by light microscopy. Representative images are shown from observations of three mice per genotype. Epididymal epithelia <i>(E)</i> and tubule lumens <i>(L)</i> are labelled. (<b>A</b>) and (<b>C</b>) show sections through wild-type epididymis showing an abundance of spermatozoa within the lumen. (<b>B</b>) and (<b>D</b>) show sections through the epididymis of <i>LKB1_SKO</i> mice showing abnormal structures within the lumen and very few spermatozoa. (<b>E</b>–<b>F</b>) TEM images of the cauda epididymal lumen from a wild-type mouse (<b>E</b>) and a <i>LKB1_SKO</i> mouse (<b>F</b>). The wild-type section shows numerous cross-sections through sperm heads (<i>N</i>) and tails (<i>arrows</i>). The section from a <i>LKB1_SKO</i> mouse shows dense luminal fluid (as indicated by the darker background to WT), cellular debris, abnormal round structures (<i>asterisk</i>) and an absence of recognisable spermatozoa cross sections (A and B, scale bar  = 20 µm; C and D, scale bar  = 50 µm; E and F, scale bar  = 5 µm).</p

Mating Assays.

<p>Mating Assays.</p

Expression of AMPK and AMPK-related kinases in testis.

<p>The mRNA expression levels of LKB1 and downstream kinases in developing testis at post-partum days 16–100. Values are shown relative to the expression levels at day 16 and shown as the mean +/- SEM, n = 5.</p

LM images showing ‘failure of spermiation’ in <i>LKB1_SKO</i> mice.

<p>Representative images of seminiferous tubules are shown at stage VIII (<b>A,B</b>), when spermiation normally occurs; and stages IX, X, and XI (<b>C</b>–<b>H</b>), after spermiation has normally taken place. The stage numbers are shown to the right of the images. Elongated spermatids are identifiable by their darkly-staining, condensed nuclei. The nuclei of immature round spermatids (<i>open arrows</i>) and elongating spermatids (<i>closed arrows</i>) are less deeply stained and can be seen embedded within the epithelium at the relevant stages of both WT and LKB1_SKO sections. There is a progressive condensation and elongation of the nucleus of the elongating spermatids from stage IX to stage XI. Sections from wild-type mice are displayed on the left. At stage VIII, elongated spermatids (<i>examples circled</i>) and cytoplasmic lobes <i>(CL)</i> are visible around the lumen. There are no elongated spermatids present around the lumen after stage VIII in wild-type sections. In contrast, in tubules from <i>LKB1_SKO</i> mice, shown on the right, elongated spermatids are visible around the lumen at all stages displayed (<i>examples circled</i>). In addition, abnormal deeply staining cytoplasmic bodies <i>(CB)</i> can be seen around the lumen, (scale bar  = 20 µm).</p

LKB1 splice variant relative gene expression and testicular localisation.

<p><b>A)</b> At day 16, prior to meiosis, testicular LKB1 is expressed at relatively low levels, with LKB1 protein below the detectable limits of colourimetric immunohistochemistry. <b>B</b>) At day 21 total LKB1 expression has increased, predominantly through increased expression of LKB1_S. LKB1 protein can be detected in meiotic spermatocytes (inset and arrow), and post-meiotic spermatids (arrowhead). At day 35 (<b>C</b>) and day 100 (<b>D</b>), the predominant transcript is LKB1_S with LKB1 protein localised to the cytoplasm of elongated spermatids in addition to round spermatids and spermatocytes, (scale bar  = 50 µm). The insert labelled ‘neg’ shows a negative control in which no primary antibody was incubated with the tissue.</p

Activity of AMPK and AMPK-related kinases in <i>LKB1_SKO</i> testis.

<p>Activity measured in immune-complexes using antibodies specific for AMPK and AMPK-related proteins are plotted as a percentage of the activity measured in wild type testis and shown as the mean±S.E.M. from three individual mice. * indicates a statistically significant difference in activity in <i>LKB1_SKO</i> compared to wild-type samples (<i>p</i><0.05 with Student's unpaired t test).</p