Host-Microbe-Drug-Nutrient Screen Identifies Bacterial Effectors of Metformin Therapy.

Metformin is the first-line therapy for treating type 2 diabetes and a promising anti-aging drug. We set out to address the fundamental question of how gut microbes and nutrition, key regulators of host physiology, affect the effects of metformin. Combining two tractable genetic models, the bacterium E. coli and the nematode C. elegans, we developed a high-throughput four-way screen to define the underlying host-microbe-drug-nutrient interactions. We show that microbes integrate cues from metformin and the diet through the phosphotransferase signaling pathway that converges on the transcriptional regulator Crp. A detailed experimental characterization of metformin effects downstream of Crp in combination with metabolic modeling of the microbiota in metformin-treated type 2 diabetic patients predicts the production of microbial agmatine, a regulator of metformin effects on host lipid metabolism and lifespan. Our high-throughput screening platform paves the way for identifying exploitable drug-nutrient-microbiome interactions to improve host health and longevity through targeted microbiome therapies. VIDEO ABSTRACT

Coupling of Rigor Mortis and Intestinal Necrosis during C. elegans Organismal Death

Organismal death is a process of systemic collapse whose mechanisms are less well understood than those of cell death. We previously reported that death in C. elegans is accompanied by a calcium-propagated wave of intestinal necrosis, marked by a wave of blue autofluorescence (death fluorescence). Here, we describe another feature of organismal death, a wave of body wall muscle contraction, or death contraction (DC). This phenomenon is accompanied by a wave of intramuscular Ca2+release and, subsequently, of intestinal necrosis. Correlation of directions of the DC and intestinal necrosis waves implies coupling of these death processes. Long-lived insulin/IGF-1-signaling mutants show reduced DC and delayed intestinal necrosis, suggesting possible resistance to organismal death. DC resembles mammalian rigor mortis, a postmortem necrosis-related process in which Ca2+influx promotes muscle hyper-contraction. In contrast to mammals, DC is an early rather than a late event in C. elegans organismal death. VIDEO ABSTRACT

Anthranilate fluorescence marks a calcium-propagated necrotic wave that promotes organismal death in C. elegans

For cells the passage from life to death can involve a regulated, programmed transition. In contrast to cell death, the mechanisms of systemic collapse underlying organismal death remain poorly understood. Here we present evidence of a cascade of cell death involving the calpain-cathepsin necrosis pathway that can drive organismal death in Caenorhabditis elegans. We report that organismal death is accompanied by a burst of intense blue fluorescence, generated within intestinal cells by the necrotic cell death pathway. Such death fluorescence marks an anterior to posterior wave of intestinal cell death that is accompanied by cytosolic acidosis. This wave is propagated via the innexin INX-16, likely by calcium influx. Notably, inhibition of systemic necrosis can delay stress-induced death. We also identify the source of the blue fluorescence, initially present in intestinal lysosome-related organelles (gut granules), as anthranilic acid glucosyl esters--not, as previously surmised, the damage product lipofuscin. Anthranilic acid is derived from tryptophan by action of the kynurenine pathway. These findings reveal a central mechanism of organismal death in C. elegans that is related to necrotic propagation in mammals--e.g., in excitotoxicity and ischemia-induced neurodegeneration. Endogenous anthranilate fluorescence renders visible the spatio-temporal dynamics of C. elegans organismal death

The spread of DF is dependent on calcium, and is accompanied by cytosolic acidosis.

<p>(A and B) <i>inx-16(ox144)</i> reduces DF and prevents its propagation (death induced using heated wire). (C–E) Ca²⁺ levels and pH in the intestine of worms killed by oxidative stress (<i>t</i>-BOOH). (C) <i>In vivo</i> Ca²⁺ levels rise at death in the anterior intestine prior to the posterior intestine, consistent with an anterior to posterior Ca²⁺ wave. Mean ± SD. (D) The Ca²⁺ reporter expressed in an <i>inx-16(ox144)</i> strain confirms that Ca²⁺, like DF, rises in the anterior but does not spread. Mean ± SD. (E) <i>In vivo</i> pH decreases at death from pH ∼7.35 to ∼6.6 in the anterior intestine prior to the posterior intestine, consistent with an anterior to posterior wave of cytosolic acidosis. Mean ± SD. In (C–E), “anterior” indicates the int1 and int2 anterior intestinal cells, and “posterior” the int9 posterior intestinal cells.</p

Characteristics of DF.

<p>(A) DF induced by hot pick killing in young WT adult (<i>N</i> = 87), L4 (<i>N</i> = 26), and male <i>C. elegans</i> (<i>N</i> = 25), and in other nematode species <i>C. briggsae</i> (<i>N</i> = 26) and <i>P. pacificus</i> (<i>N</i> = 49). ± SD, ***<i>p</i><0.001. L4 larvae and adult males show smaller increases in DF than adult hermaphrodites, perhaps due to their smaller size. (B) Typical fluorescence increase in young adult worm killed by a hot pick. (C) Fluorescence increases in an anterior to posterior wave. Figure shows mean fluorescence intensity (values normalized to highest value in each individual) at 5 points along the intestine of animals dying of old age (<i>N</i> = 29). Due to individual variation (range, 1 h 55 min–5 h 30 min, mean ∼3 h), the duration of each observation was divided into 5 equal points for measurement. T = 0 is the point at which time-lapse photography of dying (late stage C) worms was initiated. (D) DF will not propagate in a posterior to anterior wave. Head, int1 and int2 anterior intestinal cells; tail, int9 posterior intestinal cells.</p

Molecular damage does not increase blue fluorescence.

<p>(A, B) Fluorescent gut granules (arrow heads) in intestinal cells of healthy, young adult <i>C. elegans</i>. Bar, 50 µm. (C–F) Hyperoxia (5-d exposure) and free iron (1-d exposure) increase protein oxidation but not blue fluorescence, mean of 3 biological replicates ± SEM, * <i>p</i><0.05, ** <i>p</i><0.01.</p

Inhibition of necrosis pathway reduces DF.

<p>(A) The calpain-cathepsin necrosis cascade. (B) Effects of inhibition of necrosis on DF, in ER calcium mutants, a calpain mutant, lysosomal acidosis mutants, cathepsin mutants, and an innexin mutant. Death was induced by freeze-thaw in young adults. Plots in (B) and (C) show mean ± SD, *<i>p</i><0.05, **<i>p</i><0.01 and ***<i>p</i><0.001. Statistics: Wild type, <i>N</i> = 7 independent assays (different days), 50 worms/assay <i>n</i> = 350; <i>asp-4</i>, <i>N</i> = 7, <i>n</i> = 350, <i>p</i><0.001; <i>crt-1</i>, <i>N</i> = 3, <i>n</i> = 150, <i>p</i><0.01; <i>unc-68</i>, <i>N</i> = 6, <i>n</i> = 300, <i>p</i><0.001; <i>itr-1</i>, <i>N</i> = 6, <i>n</i> = 300, <i>p</i><0.01; <i>tra-3</i>, <i>N</i> = 6, <i>n</i> = 300, <i>p</i><0.001; <i>vha-12</i>, <i>N</i> = 7, <i>n</i> = 350, <i>p</i>>0.05; <i>unc-32</i>, <i>N</i> = 6, <i>n</i> = 300, <i>p</i>>0.05; <i>cad-1</i>, <i>N</i> = 7, <i>n</i> = 350, <i>p</i><0.001; <i>inx-16</i>, <i>N</i> = 6, <i>n</i> = 300, <i>p</i><0.001. (C) Most necrosis mutants are significantly more resistant to death induced by osmotic stress (500 mM NaCl). Wild type, <i>N</i> = 6, <i>n</i> = 300; <i>asp-4</i>, <i>N</i> = 7, <i>n</i> = 350, <i>p</i>>0.05; <i>crt-1</i>, <i>N</i> = 5, <i>n</i> = 250, <i>p</i>>0.05; <i>unc-68</i>, <i>N</i> = 2, <i>n</i> = 150, <i>p</i>>0.05; <i>itr-1</i>, <i>N</i> = 3, <i>n</i> = 300, <i>p</i><0.001; <i>tra-3</i>, <i>N</i> = 3, <i>n</i> = 100, <i>p</i><0.01; <i>vha-12</i>, <i>N</i> = 3, <i>n</i> = 150, <i>p</i>>0.05; <i>unc-32</i>, <i>N</i> = 2, <i>n</i> = 100, <i>p</i><0.05; <i>cad-1</i>, <i>N</i> = 6, <i>n</i> = 300, <i>p</i><0.001; <i>inx-16</i>, <i>N</i> = 4, <i>n</i> = 200, <i>p</i><0.001.</p

Repurposing metformin: an old drug with new tricks in its binding pockets

Improvements in healthcare and nutrition have generated remarkable increases in life expectancy worldwide. This is one of the greatest achievements of the modern world yet it also presents a grave challenge: as more people survive into later life, more also experience the diseases of old age, including type 2 diabetes (T2D), cardiovascular disease (CVD) and cancer. Developing new ways to improve health in the elderly is therefore a top priority for biomedical research. Although our understanding of the molecular basis of these morbidities has advanced rapidly, effective novel treatments are still lacking. Alternative drug development strategies are now being explored, such as the repurposing of existing drugs used to treat other diseases. This can save a considerable amount of time and money since the pharmacokinetics, pharmacodynamics and safety profiles of these drugs are already established, effectively enabling preclinical studies to be bypassed. Metformin is one such drug currently being investigated for novel applications. The present review provides a thorough and detailed account of our current understanding of the molecular pharmacology and signalling mechanisms underlying biguanide–protein interactions. It also focuses on the key role of the microbiota in regulating age-associated morbidities and a potential role for metformin to modulate its function. Research in this area holds the key to solving many of the mysteries of our current understanding of drug action and concerted effects to provide sustained and long-life health