a b s t r a c t GPR41 is reportedly expressed in murine adipose tissue and mediates short chain fatty acid (SCFA)-stimulated leptin secretion by activating Ga i . Here, we agree with a contradictory report in finding no expression of GPR41 in murine adipose tissue. Nevertheless, in the presence of adenosine deaminase to minimise Ga i signalling via the adenosine A1 receptor, SCFA stimulated leptin secretion by adipocytes from wild-type but not GPR41 knockout mice. Expression of GPR43 was reduced in GPR41 knockout mice. Acetate but not butyrate stimulated leptin secretion in wild-type mesenteric adipocytes, consistent with mediation of the response by GPR43 rather than GPR41. Pertussis toxin prevented stimulation of leptin secretion by propionate in epididymal adipocytes, implicating Ga i signalling mediated by GPR43 in SCFA-stimulated leptin secretion.

SummaryThe citric acid cycle (CAC) metabolite fumarate has been proposed to be cardioprotective; however, its mechanisms of action remain to be determined. To augment cardiac fumarate levels and to assess fumarate's cardioprotective properties, we generated fumarate hydratase (Fh1) cardiac knockout (KO) mice. These fumarate-replete hearts were robustly protected from ischemia-reperfusion injury (I/R). To compensate for the loss of Fh1 activity, KO hearts maintain ATP levels in part by channeling amino acids into the CAC. In addition, by stabilizing the transcriptional regulator Nrf2, Fh1 KO hearts upregulate protective antioxidant response element genes. Supporting the importance of the latter mechanism, clinically relevant doses of dimethylfumarate upregulated Nrf2 and its target genes, hence protecting control hearts, but failed to similarly protect Nrf2-KO hearts in an in vivo model of myocardial infarction. We propose that clinically established fumarate derivatives activate the Nrf2 pathway and are readily testable cytoprotective agents.

Rationale: AMP-activated protein kinase (AMPK) is an important regulator of energy balance and signaling in the heart. Mutations affecting the regulatory ␥2 subunit have been shown to cause an essentially cardiacrestricted phenotype of hypertrophy and conduction disease, suggesting a specific role for this subunit in the heart.Objective: The ␥ isoforms are highly conserved at their C-termini but have unique N-terminal sequences, and we hypothesized that the N-terminus of ␥2 may be involved in conferring substrate specificity or in determining intracellular localization. Methods and Results:A yeast 2-hybrid screen of a human heart cDNA library using the N-terminal 273 residues of ␥2 as bait identified cardiac troponin I (cTnI) as a putative interactor. In vitro studies showed that cTnI is a good AMPK substrate and that Ser150 is the principal residue phosphorylated. Furthermore, on AMPK activation during ischemia, Ser150 is phosphorylated in whole hearts. Using phosphomimics, measurements of actomyosin ATPase in vitro and force generation in demembraneated trabeculae showed that modification at Ser150 resulted in increased Ca 2؉ Key Words: familial hypertrophic cardiomyopathy Ⅲ myocardial contractility Ⅲ phosphorylation A MP-activated protein kinase (AMPK) is a crucial component of a highly conserved serine/threonine protein kinase cascade central to the control of energy balance at the cellular and whole-body levels. 1,2 AMPK exists as a ␣␤␥ heterotrimer, with ␣ being the catalytic subunit, and the ␤ and ␥ subunits performing structural and regulatory functions. Isoforms of all subunits have been identified (␣1, ␣2, ␤1, ␤2, ␥1, ␥2, and ␥3), each being encoded by a different gene (PRKAA1, PRKAA2, PRKAB1, PRKAB2, PRKAG1, PRKAG2, and PRKAG3, respectively). The ␣ subunits consist of a typical serine/threonine protein kinase domain at the N-terminus (which also contains the critical phosphorylation site for AMPK activation, Thr172 3 ) and a C-terminal domain involved in the binding of the ␤ and ␥ subunits. 1,2 The ␤ subunits are myristoylated at their N-terminus, contain a conserved C-terminal domain that is involved in binding of the ␣ and ␥ subunits, and a carbohydrate binding domain. The carbohydrate binding domain may allow AMPK to sense the status of cellular energy reserves in the form of glycogen in addition to responding to AMP/ATP levels. 4 The ␥ subunits have a high degree of homology in their C-terminal Original received October 31, 2011; revision received March 14, 2012; accepted March 19, 2012. In February 2012 sequences, all containing 2 pairs of highly conserved cystathionine ␤-synthase domains, which have been shown to be directly involved in the binding of adenine nucleotides. [5][6][7] In contrast, their N-terminal regions are highly variable, with ␥2 and ␥3 possessing different long N-terminal extensions compared with the shorter ␥1 isoform (Figure 1). The ␥2 and ␥3 N-terminal sequences appear to be unique in that they do not share sequence identity with each other nor with any known protein. ...

SummaryDespite significant advances in our understanding of the biology determining systemic energy homeostasis, the treatment of obesity remains a medical challenge. Activation of AMP-activated protein kinase (AMPK) has been proposed as an attractive strategy for the treatment of obesity and its complications. AMPK is a conserved, ubiquitously expressed, heterotrimeric serine/threonine kinase whose short-term activation has multiple beneficial metabolic effects. Whether these translate into long-term benefits for obesity and its complications is unknown. Here, we observe that mice with chronic AMPK activation, resulting from mutation of the AMPK γ2 subunit, exhibit ghrelin signaling-dependent hyperphagia, obesity, and impaired pancreatic islet insulin secretion. Humans bearing the homologous mutation manifest a congruent phenotype. Our studies highlight that long-term AMPK activation throughout all tissues can have adverse metabolic consequences, with implications for pharmacological strategies seeking to chronically activate AMPK systemically to treat metabolic disease.

SCFA are produced in the gut by bacterial fermentation of undigested carbohydrates. Activation of the Ga i -protein-coupled receptor GPR41 by SCFA in b-cells and sympathetic ganglia inhibits insulin secretion and increases sympathetic outflow, respectively. A possible role in stimulating leptin secretion by adipocytes is disputed. In the present study, we investigated energy balance and glucose homoeostasis in GPR41 knockout mice fed on a standard low-fat or a high-fat diet. When fed on the low-fat diet, body fat mass was raised and glucose tolerance was impaired in male but not female knockout mice compared to wild-type mice. Soleus muscle and heart weights were reduced in the male mice, but total body lean mass was unchanged. When fed on the high-fat diet, body fat mass was raised in male but not female GPR41 knockout mice, but by no more in the males than when they were fed on the low-fat diet. Body lean mass and energy expenditure were reduced in male mice but not in female knockout mice. These results suggest that the absence of GPR41 increases body fat content in male mice. Gut-derived SCFA may raise energy expenditure and help to protect against obesity by activating GPR41.

Highlights d Human iPSC-cardiomyocytes were used for MAP4K4 target validation and drug discovery d MAP4K4 shRNA protects hiPSC-cardiomyocytes from lethal oxidative stress d MAP4K4 inhibitors promote hiPSC-cardiomyocyte survival and function d MAP4K4 inhibition markedly reduces cardiac ischemiareperfusion injury in mice

AMP-activated protein kinase (AMPK) plays diverse roles and coordinates complex metabolic pathways for maintenance of energy homeostasis. This could be explained by the fact that AMPK exists as multiple heterotrimer complexes comprising a catalytic α-subunit (α1 and α2) and regulatory β (β1 and β2)- and γ (γ1, γ2, γ3)-subunits, which are uniquely distributed across different cell types. There has been keen interest in developing specific and isoform-selective AMPK-activating drugs for therapeutic use and also as research tools. Moreover, establishing ways of enhancing cellular AMPK activity would be beneficial for both purposes. Here, we investigated if a recently described potent AMPK activator called 991, in combination with the commonly used activator 5-aminoimidazole-4-carboxamide riboside or contraction, further enhances AMPK activity and glucose transport in mouse skeletal muscle ex vivo. Given that the γ3-subunit is exclusively expressed in skeletal muscle and has been implicated in contraction-induced glucose transport, we measured the activity of AMPKγ3 as well as ubiquitously expressed γ1-containing complexes. We initially validated the specificity of the antibodies for the assessment of isoform-specific AMPK activity using AMPK-deficient mouse models. We observed that a low dose of 991 (5 μM) stimulated a modest or negligible activity of both γ1- and γ3-containing AMPK complexes. Strikingly, dual treatment with 991 and 5-aminoimidazole-4-carboxamide riboside or 991 and contraction profoundly enhanced AMPKγ1/γ3 complex activation and glucose transport compared with any of the single treatments. The study demonstrates the utility of a dual activator approach to achieve a greater activation of AMPK and downstream physiological responses in various cell types, including skeletal muscle.

No abstract