Targeting ERK3/MK5 complex for treatment of obesity and diabetes.

Kinases represent one of the largest druggable families of proteins. Importantly, many kinases are aberrantly activated/de-activated in multiple organs during obesity, which contributes to the development of diabetes and associated diseases. Previous results indicate that the complex between Extracellular-regulated kinase 3 (ERK3) and Mitogen-Activated Protein Kinase (MAPK)-activated protein kinase 5 (MK5) suppresses energy dissipation and promotes fatty acids (FAs) output in adipose tissue and, therefore promotes obesity and diabetes. However, the therapeutic potential of targeting this complex at the systemic level has not been fully explored. Here we applied a translational approach to target the ERK3/MK5 complex in mice. Importantly, deletion of ERK3 in the whole body or administration of MK5-specific inhibitor protects against obesity and promotes insulin sensitivity. Finally, we show that the expression of ERK3 and MK5 correlates with the degree of obesity and that ERK3/MK5 complex regulates energy dissipation in human adipocytes. Altogether, we demonstrate that ERK3/MK5 complex can be targeted in vivo to preserve metabolic health and combat obesity and diabetes.This study was funded by European Research Council (ERC) Starting Grant SicMetabol (no.678119), Emmy Noether Grant Su820/1-1 from the German Research Foundation (DFG), EMBO Installation Grant from European Molecular Biology Organization (EMBO), the Dioscuri Centre of Scientific Excellenced The program initiated by the Max Planck Society (MPG), managed jointly with the National Science Centre, and mutually funded by the Ministry of Science and Higher Education (MNiSW) and the German Federal Ministry of Education and Research (BMBF), and Sonata bis grant (2020/38/E/NZ4/00314) from National Science Centre.S

Die Proteinkinase D2 treibt den Chylomicron-vermittelten Lipidtransport im Darm an und fördert Fettleibigkeit

Obesity and associated metabolic syndrome are growing concerns in modern society due to the negative consequences for human health and well-being. Cardiovascular diseases and type 2 diabetes are only some of the pathologies associated to overweight. Among the main causes are decreased physical activity and food availability and composition. Diets with high content of fat are energy-dense and their overconsumption leads to an energy imbalance, which ultimately promotes energy storage as fat and obesity. Aberrant activation of signalling cascades and hormonal imbalances are characteristic of this disease and members of the Protein Kinase D (PKD) family have been found to be involved in several mechanisms mediating metabolic homeostasis. Therefore, we aimed to investigate the role of Protein Kinase D2 (PKD2) in the regulation of metabolism. Our investigation initiated with a mice model for global PKD2 inactivation, which allowed us to prove a direct involvement of this kinase in lipids homeostasis and obesity. Inactivation of PKD2 protected the mice from high-fat diet-induced obesity and improved their response to glucose, insulin and lipids. Furthermore, the results indicated that, even though there were no changes in energy intake or expenditure, inactivation of PKD2 limited the absorption of fat from the intestine and promoted energy excretion in feces. These results were verified in a mice model for specific deletion of intestinal PKD2. These mice not only displayed an improved metabolic fitness but also a healthier gut microbiome profile. In addition, we made use of a small-molecule inhibitor of PKD in order to prove that local inhibition of PKD2 in the intestine was sufficient to inhibit lipid absorption. The usage of the inhibitor not only protected the mice from obesity but also was efficient in avoiding additional body-weight gain after obesity was pre-established in mice. Mechanistically, we determined that PKD2 regulates lipids uptake in enterocytes by phosphorylation of Apolipoprotein A4 (APOA4) and regulation of chylomicron-mediated triglyceride absorption. PKD2 deletion or inactivation increased abundance of APOA4 and decreased the size of chylomicrons and therefore lipids absorption from the diet. Moreover, intestinal activation of PKD2 in human obese patients correlated with higher levels of triglycerides in circulation and a detrimental blood profile. In conclusion, we demonstrated that PKD2 is a key regulator of dietary fat absorption in murine and human context, and its inhibition might contribute to the treatment of obesity.Fettleibigkeit und das damit verbundene metabolische Syndrom stellt in der modernen Gesellschaft aufgrund der negativen Folgen für die menschliche Gesundheit und das Wohlbefinden ein zunehmendes Problem dar. Herz-Kreislauf-Erkrankungen und Typ- 2-Diabetes sind nur einige der mit Übergewicht verbundenen Pathologien. Zu den Hauptursachen zählen eine verminderte körperliche Aktivität sowie die Verfügbarkeit und Zusammensetzung von Nahrungsmitteln. Diäten mit hohem Fettgehalt haben eine hohe Energiedichte und ihr übermäßiger Konsum führt zu einem Energieungleichgewicht, das letztendlich die Energiespeicherung als Fett und Fettleibigkeit fördert. Aberrante Aktivierung von Signalkaskaden und hormonelle Ungleichgewichte sind charakteristisch für diese Krankheit, und es wurde festgestellt, dass Mitglieder der Protein Kinase D (PKD) -Familie an mehreren Mechanismen der metabolischen Homöostase beteiligt sind. Daher zielten wir darauf ab die Rolle der Proteinkinase D2 (PKD2) bei der Regulation des Stoffwechsels zu untersuchen. Unsere Untersuchung begann mit einem Mäusemodell für die globale PKD2- Inaktivierung, welches es uns ermöglichte, eine direkte Beteiligung dieser Kinase an der Lipidhomöostase und Fettleibigkeit nachzuweisen. Die Inaktivierung von PKD2 schützte die Mäuse vor fettreicher diätbedingter Fettleibigkeit und verbesserte ihre Reaktion auf Glukose, Insulin und Lipide. Darüber hinaus zeigten die Ergebnisse, dass die Inaktivierung von PKD2 die Absorption von Fett über den Darm begrenzte und die Energieausscheidung im Kot förderte, obwohl sich die Energieaufnahme oder der Energieverbrauch nicht änderten. Diese Ergebnisse wurden in einem Mäusemodell mit spezifischer Deletion von intestinaler PKD2 verifiziert. Diese Mäuse zeigten nicht nur eine verbesserte metabolische Fitness, sondern auch ein gesünderes Darmmikrobiomprofil. Zusätzlich verwendeten wir einen niedermolekularen PKD- Inhibitor, um zu beweisen, dass die lokale Hemmung von PKD2 im Darm ausreicht, um die Lipidabsorption zu hemmen. Die Verwendung des Inhibitors schützte die Mäuse nicht nur vor Fettleibigkeit, sondern verhinderte auch wirksam eine zusätzliche Gewichtszunahme, nachdem bei Mäusen bereits Fettleibigkeit festgestellt worden war. Mechanistisch haben wir festgestellt, dass PKD2 die Lipidaufnahme in Enterozyten durch Phosphorylierung von Apolipoprotein A4 (APOA4) und Regulation der Chylomicron-vermittelten Triglyceridabsorption reguliert. Die Deletion oder Inaktivierung von PKD2 erhöhte die Häufigkeit von APOA4 und verringerte die Größe der Chylomikronen und damit die Lipidabsorption aus der Nahrung. Darüber hinaus korrelierte die intestinale Aktivierung von PKD2 bei adipösen Patienten mit höheren Triglyceridspiegeln im Kreislauf und einem schädlichen Blutprofil. Zusammenfassend haben wir gezeigt, dass PKD2 ein Schlüsselregulator für die Aufnahme von Nahrungsfett im murinen und menschlichen Kontext ist und seine Hemmung zur Behandlung von Fettleibigkeit beitragen könnt

Phospholipases D1 and D2 Suppress Appetite and Protect against Overweight

<div><p>Obesity is a major risk factor predisposing to the development of peripheral insulin resistance and type 2 diabetes (T2D). Elevated food intake and/or decreased energy expenditure promotes body weight gain and acquisition of adipose tissue. Number of studies implicated phospholipase D (PLD) enzymes and their product, phosphatidic acid (PA), in regulation of signaling cascades controlling energy intake, energy dissipation and metabolic homeostasis. However, the impact of PLD enzymes on regulation of metabolism has not been directly determined so far. In this study we utilized mice deficient for two major PLD isoforms, PLD1 and PLD2, to assess the impact of these enzymes on regulation of metabolic homeostasis. We showed that mice lacking PLD1 or PLD2 consume more food than corresponding control animals. Moreover, mice deficient for PLD2, but not PLD1, present reduced energy expenditure. In addition, deletion of either of the PLD enzymes resulted in development of elevated body weight and increased adipose tissue content in aged animals. Consistent with the fact that elevated content of adipose tissue predisposes to the development of hyperlipidemia and insulin resistance, characteristic for the pre-diabetic state, we observed that <i>Pld1</i>^<i>-/-</i> and <i>Pld2</i>^<i>-/-</i> mice present elevated free fatty acids (FFA) levels and are insulin as well as glucose intolerant. In conclusion, our data suggest that deficiency of PLD1 or PLD2 activity promotes development of overweight and diabetes.</p></div

Deletion of <i>Pld1</i> or <i>Pld2</i> does not affect satiety response.

<p>A) 24 hours cumulative food intake of 20-weeks old mice with indicated genotypes. B) Food intake at different time-points after overnight fasting of 20-weeks old mice with indicated genotypes. Data represented as mean +/- S.E.M., n = 6 females for control (black), n = 4 females for <i>Pld1</i>^<i>-/-</i> (orange), n = 6 females for <i>Pld2</i>^<i>-/-</i> (green). *p<0.05, **p<0.01, ***p<0.001</p

mRNA expression in hypothalamus of neuropeptides controlling food intake.

<p>Relative expression of mRNA in hypothalamus of <i>Pld1</i>^<i>-/-</i>, <i>Pld2</i>^<i>-/-</i> and control mice. The analyzed targets are presented as those with known orexigenic effect: neuropeptide Y (Npy), neuropeptide Y receptor 1 (Npyr1), Agouti Related Neuropeptide (AgRp), Hypocretin (Hcrt), Galanin (Gal); those with anorexigenic effect: Pro-opiomelanocortin (Pomc), Cocaine-amphetamine-regulated transcript (Cart), Corticotropin-releasing factor (Crf), Neuromedin U (Nmu); and those involved in the metabolism of GABA and glutamate: Glutamate-ammonia ligase (Glul), Glutamate decarboxylase (Gad1), Glutaminase (Gls) and 4-aminobutyrate aminotransferase (Abat). Data represented as mean +/- S.E.M., n = 6 males for control (black), n = 5 males for <i>Pld1</i>^<i>-/-</i> (orange), n = 6 males for <i>Pld2</i>^<i>-/-</i> (green). *p<0.05, **p<0.01, ***p<0.001</p

Deletion of <i>Pld1</i> or <i>Pld2</i> promotes food intake.

<p>A) 24 hours cumulative food intake of 20-weeks old mice with indicated genotypes. B) Oxygen consumption of mice with indicated genotypes at different time of the day. C) Average oxygen consumption during light and dark phase. D) Carbon dioxide production of mice with indicated genotypes at different time of the day. E) Average carbon dioxide during light and dark phase. F) Voluntary movements of mice with indicated genotypes at different time of the day. G) Average voluntary movements of mice during light and dark phase. H) Respiratory exchange rate of mice with indicated genotypes at different time of the day. I) Average respiratory exchange rate of mice during light and dark phase. Data represented as mean +/- S.E.M., n = 6 males for control (black), n = 4 males for <i>Pld1</i>^<i>-/-</i> (orange), n = 6 males for <i>Pld2</i>^<i>-/-</i> (green). *p<0.05, **p<0.01, ***p<0.001</p

Free fatty acids in circulation are elevated in mice deficient for PLD1 or PLD2.

<p>A) Free fatty acids (FFAs), B) Glycerol, C) Triglycerides in circulation of 18-weeks old mice with indicated genotypes. Data represented as mean +/- S.E.M., n = 9 males for control (black), n = 6 males for <i>Pld1</i>^<i>-/-</i> (orange), n = 8 males for <i>Pld2</i>^<i>-/-</i> (green). *p<0.05, **p<0.01, ***p<0.001</p

Deletion of Pld1 or Pld2 promotes insulin resistance and glucose intolerance.

<p>A) Insulin tolerance test in <i>Pld1</i>^<i>-/-</i>, <i>Pld2</i>^<i>-/-</i> and control mice at 16 weeks old. n = 7 for control (black), n = 6 for <i>Pld1</i>^<i>-/-</i> (orange), n = 8 for <i>Pld2</i>^<i>-/-</i> (green). B) Area under the curve for the insulin tolerance test. C) Glucose tolerance test in <i>Pld1</i>^<i>-/-</i>, <i>Pld2</i>^<i>-/-</i> and control mice at 18 weeks old. n = 12 for control (black), n = 10 for <i>Pld1</i>^<i>-/-</i> (orange), n = 12 for <i>Pld2</i>^<i>-/-</i> (green). D) Area under the curve for the glucose tolerance test. E) Insulin levels in circulation of mice with indicated genotypes at 20 weeks old. n = 8 males for control (black), n = 6 males for <i>Pld1</i>^<i>-/-</i> (orange), n = 8 males for <i>Pld2</i>^<i>-/-</i> (green). Data represented as mean +/- S.E.M., *p<0.05, **p<0.01, ***p<0.001</p

The kinase PKD3 provides negative feedback on cholesterol and triglyceride synthesis by suppressing insulin signaling

Hepatic activation of protein kinase C (PKC) isoforms by diacylglycerol (DAG) promotes insulin resistance and contributes to the development of type 2 diabetes (T2D). The closely related protein kinase D (PKD) isoforms act as effectors for DAG and PKC. Here, we showed that PKD3 was the predominant PKD isoform expressed in hepatocytes and was activated by lipid overload. PKD3 suppressed the activity of downstream insulin effectors including the kinase AKT and mechanistic target of rapamycin complex 1 and 2 (mTORC1 and mTORC2). Hepatic deletion of PKD3 in mice improved insulin-induced glucose tolerance. However, increased insulin signaling in the absence of PKD3 promoted lipogenesis mediated by SREBP (sterol regulatory element-binding protein) and consequently increased triglyceride and cholesterol content in the livers of PKD3-deficient mice fed a high-fat diet. Conversely, hepatic-specific overexpression of a constitutively active PKD3 mutant suppressed insulin-induced signaling and caused insulin resistance. Our results indicate that PKD3 provides feedback on hepatic lipid production and suppresses insulin signaling. Therefore, manipulation of PKD3 activity could be used to decrease hepatic lipid content or improve hepatic insulin sensitivity

Targeting ERK3/MK5 complex for treatment of obesity and diabetes.

This dataset corresponds to the article titled “Targeting ERK3/MK5 complex for treatment of obesity and diabetes” published in Biochem Biophys Res Commun. 2022 Jul 5;612:119-125. doi: 10.1016/j.bbrc.2022.04.070. It comprises images of the original Western blots, along with the source data used for creating figures and conducting calculations. Further information related to this dataset can be found in the correspondingly titled article. This study was funded by European Research Council (ERC) Starting Grant SicMetabol (no.678119), Emmy Noether Grant Su820/1-1 from the German Research Foundation (DFG), EMBO Installation Grant from European Molecular Biology Organization (EMBO), the Dioscuri Centre of Scientific Excellence—The program initiated by the Max Planck Society (MPG), managed jointly with the National Science Centre, and mutually funded by the Ministry of Science and Higher Education (MNiSW) and the German Federal Ministry of Education and Research (BMBF), and Sonata bis grant (2020/38/E/NZ4/00314) from National Science Centre