Use of the ppard / PDK4 / angptl4 pathway as a biomarker and biotarget of motility disorders in patients with obesity

Abstract

Obesity often causes stomach dysmotility, but the underlying mechanisms are unclear. The inventors studied the gastric smooth muscle homeostasis in patients and mice with obesity, and in human cells exposed to fatty acids. They found that fatty acids change the differentiation status of smooth muscle cells (SMCs) by activating PPARD and increasing the expression of PDK4 and ANGPTL4, two genes that mark the alteration and immaturity of gastric smooth muscle in obesity. Blocking PDK4 or ANGPTL4 prevents the fatty acid-induced changes in SMCs. The invention uses the PPARD / PDK4 / ANGPTL4 pathway as a biomarker and biotarget of motility disorders in obese patients, and provides methods and kits for their diagnosis, prognosis, monitoring, and treatment.

Description

[0001] USE OF THE PPARD / PDK4 / ANGPTL4 PATHWAY AS A BIOMARKER AND BIOTARGET OF MOTILITY DISORDERS IN PATIENTS WITH OBESITY

[0002] FIELD OF THE INVENTION:

[0003] The present invention is in the field of medicine, in particular gastroenterology.

[0004] BACKGROUND OF THE INVENTION:

[0005] Obesity, a prevalent chronic condition affecting approximately 13% of the adult population, presents a significant global public health challenge (NCD Risk Factor Collaboration, NCD- RisC, Lancet 2024). It is strongly linked with various medical conditions, including type 2 diabetes, cardiovascular diseases, and strongest evidence exists for an association of obesity with several cancer types (endometrial, postmenopausal breast, prostate, and renal) (Afshin A et al, NEJM, 2017; De Oergola and Silverstris, J of Obesity, 2013). Moreover, individuals with obesity frequently experience gastrointestinal (GI) issues including gastroesophageal reflux, nonalcoholic fatty liver disease, an elevated colorectal and esophageal adenocarcinoma cancer risk, and functional GI disorders (e.g. dyspepsia, inflammatory bowel syndrome, constipation and diarrhea) (Eslick GD 2012; Camilleri M, Acosta A 2016; Delgado- Aros et al, Am J Gastroen 2004). Despite their prevalence, the comprehensive understanding of GI complications in obesity is insufficiently explored in both clinical practice and research.

[0006] Moreover, the nature of the GI tract, characterized by its regionalization and division along the rostro-caudal axis, complicates and frequently yields contradictory findings in studies concerning obesity impacting the GI motility (de Santa Barbara et al, 2003; Steenackers et al Neurogastro 2023). Using both genetically and diet-induced obese mice, it was observed that notable changes in gene expression take place in the stomach rather than in the small intestine or colon, emphasizing the role of the stomach in obesity (Chen et al, Physiological Reports, 2016; Steenackers et al Neurogastro 2023). Obesity results from an imbalance between energy intake and expenditure, predominantly ensured by the food intake and satiety mechanism regulated by the stomach. Among then, gastric emptying is crucial for maintaining energy balance and body weight regulation (Camilari et al, 2015). In humans, several studies suggest that accelerated gastric emptying may disrupt satiety signaling, potentially leading to increased food intake and contributing to the pathophysiology of obesity and its related complications (Wright et al, Gastroen 1983; Christian et al, J Nucl Med, 1986; Camilari et al, 2017). Recent research has linked faster gastric emptying to notable weight gain in younger adults (Pajot et al, Int J Ob 2020). Gastric emptying is a complex functional mechanism that requires the generation and conduction of regular depolarizing potential (slow wave) in the smooth muscle, events initiated in the interstitial cells of Cajal (ICC) that are electrically coupled to the smooth muscle cells (SMC) (Di Natale MR, Neurogastroent Motil 2023; Ward and Sanders, AMJPhyiolGastro2001; Ordog et al, J Physio 1999; Chevalier et al, 2020). Interestingly, the stomach of early-induced obese mice harbors increased proliferation and density of ICCs, which contributes to the accelerated gastric emptying observed in these obese mice (Hayashi et al, Gastroenterology, 2017). However, the impact of obesity on the gastric smooth muscle has been undervalued, even though disturbances in its activity can result in motility issues (Herring et al, BMC Med Genomics 2019; Scirocco et al 2016, le Guen et al 2015, Martire et al 2021, Notarnicola et al 2012), possibly stemming from an inadequate grasp of GI smooth muscle cell physiology.

[0007] Digestive SMCs originate from LIX1 -positive mesenchymal progenitors through a two-step process (McKey et al, 2016; Guerin et al, 2022). These progenitors first undergo a determination program characterized by the expression of MYOCARDIN, a cofactor of the transcription factor SRF (Serum Response Factor), which controls the expression of smooth muscle actin proteins, including gamma (ySMA) and alpha (aSMA) smooth muscle actins (Wang et al, 2003). Following this, determined SMCs enter a more differentiated state, marked by cell elongation and the expression of CALPONIN1 and SM22, two actin-binding proteins involved in smooth muscle contractility (Le Guen et al, 2015). As SMCs differentiate, they acquire the capacity to generate force and contract, facilitated by the presence and organization of smooth muscle actin and myosin proteins. While skeletal muscle contains satellite cells that respond to growth and injury, a similar reservoir of cells presenting pluripotency capacities has not been identified in smooth muscles. Instead, SMCs possess the ability to dedifferentiate under stimulation, revert to a mesenchymal phenotype, and re-enter the cell-division cycle to proliferate (Le Guen et al, 2015; Brittingham J, Gastroen 1998). This physiological process is crucial during pediatric intestine smooth muscle growth (Struijs et al, J Ped Surg 2009). However, an imbalance in plasticity favoring the mesenchymal progenitor state is suspected to contribute to numerous human diseases, including primary visceral myopathy, inflammatory bowel disease, diabetes, and metabolic disorders (Martire et al 2022; Nair et al Blennerhaussett 2014). Consequently, this imbalance leads to impaired GI motility (Le Guen et al, 2015; Scirocco et al, 2016). SUMMARY OF THE INVENTION:

[0008] The present invention is defined by the claims. In particular, the present invention relates to methods determining whether patients suffering from obesity are at risk of having a motility disorder and to methods for treating said motility disorders.

[0009] DETAILED DESCRIPTION OF THE INVENTION:

[0010] Clinical research has uncovered the fact that stomach dysmotility is a common characteristic of obesity. However, the specific mechanisms driving gastric emptying dysfunction in patients with obesity remain largely unidentified. In the present invention, the inventors investigated potential mechanisms by focusing on the homeostasis of gastric smooth muscle, utilizing both patient and mice tissue samples, and human cell culture system. In particular, the inventors showed that the smooth muscle layers in the gastric tissue of both patients with obesity and mice on a High-Fat Diet show changes in their differentiation status. Treating cultured human gastric SMCs with fatty acids mimic these alterations and are linked to the stimulation of PDK4 and ANGPTL4 expression. Preventing the upregulation of PDK4 or ANGPTL4 expression inhibits the lipid-induced changes in SMCs. Lipid treatment stimulates PPARD activity, its nuclear localization and consequently the mRNA expressions of PDK4 and ANGPTL4 leading to SMC dedifferentiation.. The expression of PDK4 and ANGPTL4 correlates with the alteration and immaturity of gastric smooth muscle in patients with obesity. In conclusion, obesity triggers a specific immaturity in the gastric smooth muscle, driven by the activation of the PPARD / PDK4 / ANGPTL4 pathway. These novel mechanistic findings offer potential for developing biomarkers to identify stomach dysmotility in patients with obesity.

[0011] Accordingly, the first object of the present invention relates to a method of determining whether a patient suffering from obesity is at risk of having a motility disorder comprising the step consisting of determining the level of PDK4 and / or ANGPTL4 in a sample obtained from the patient wherein said level correlates with the risk of having a motility disorder.

[0012] As used herein, the term "subject", “host”, “individual” or “patient” refers to a mammal, typically a human being, male or female at any age.

[0013] As used herein the term "obesity" refers to a condition characterized by an excess of body fat. The operational definition of obesity is based on the Body Mass Index (BMI), which is calculated as body weight per height in squared meter (kg / m2). Obesity refers to a condition whereby an otherwise healthy patient has a BMI greater than or equal to 30 kg / m2, or a condition whereby a patient with at least one co-morbidity has a BMI greater than or equal to 27 kg / m2. An "obese patient" is an otherwise patient with a BMI greater than or equal to 30 kg / m2or a patient with at least one co-morbidity with a BMI greater than or equal 27 kg / m2. A "patient at risk of obesity" is an otherwise healthy patient with a BMI of 25 kg / m2to less than 30 kg / m2or a patient with at least one co-morbidity with a BMI of 25 kg / m2to less than 27 kg / m2. The increased risks associated with obesity may occur at a lower BMI in people of Asian descent. In Asian and Asian-Pacific countries, including Japan, "obesity" refers to a condition whereby a patient with at least one obesity -induced or obesity-related co-morbidity that requires weight reduction or that would be improved by weight reduction, has a BMI greater than or equal to 25 kg / m2. An "obese patient" in these countries refers to a patient with at least one obesity- induced or obesity-related co-morbidity that requires weight reduction or that would be improved by weight reduction, with a BMI greater than or equal to 25 kg / m2. In these countries, a "patient at risk of obesity" is a person with a BMI greater than 23 kg / m2 to less than 25 kg / m2.

[0014] As used herein, the term “motility disorder” or “dysmotility” has its general in the art and refers to a condition in which muscles of the digestive system do not work as they should. This dysfunction can alter the speed, strength, or coordination of the esophagus, stomach, small intestine, and / or the large intestine. The term includes esophageal dysmotility, stomach dysmotility, and intestinal dysmotility.

[0015] In particular, the method of the present invention is particularly suitable for determining whether the patient is at risk of having a stomach dysmotility.

[0016] More particularly, the method of the present invention is particularly suitable for determining whether the patient who underwent bariatric surgery is at risk of having a stomach dysmotility.

[0017] As used herein, the term "bariatric surgery" refers to a variety of surgical procedures performed on individuals with severe obesity to facilitate weight reduction and improve obesity-related health conditions. These procedures typically alter the digestive system to limit food intake, absorption, or both. Common types of bariatric surgery include gastric bypass, sleeve gastrectomy, and adjustable gastric banding. The primary goal of bariatric surgery is to achieve significant and sustained weight loss, thereby reducing the risk of obesity-related comorbidities such as type 2 diabetes, hypertension, and cardiovascular disease.

[0018] As used herein, the term “stomach dysmotility” refers to a condition in which the stomach does not empty its contents normally. This can cause symptoms such as nausea, vomiting, bloating, or fullness. Stomach dysmotility can be caused by various factors, such as nerve damage, hormonal imbalance, infections, medications, or obesity. Stomach dysmotility can affect the digestion and absorption of nutrients and lead to complications such as malnutrition, dehydration, or bacterial overgrowth.

[0019] As used herein, the term "risk" in the context of the present invention, relates to the probability that a liver-related event will occur over a specific time period and can mean a subject's "absolute" risk or "relative" risk. Absolute risk can be measured with reference to either actual observation post-measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period. Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed. Odds ratios, the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p / (l-p) where p is the probability of event and (1- p) is the probability of no event) to no- conversion. "Risk evaluation," or "evaluation of risk" in the context of the present invention encompasses making a prediction of the probability, odds, or likelihood that an event may occur, the rate of occurrence of the event. Risk evaluation can also comprise prediction of future clinical parameters, traditional laboratory risk factor values, or other indices, either in absolute or relative terms in reference to a previously measured population.

[0020] As used herein, the term “sample” refers to any sample obtained from the subject for the purpose of performing the method of the present invention. The sample is used to measure the expression level of one or more biomarkers related to mobility disorder, according to the methods of the present invention. In some embodiments, the sample is a bodily fluid (e.g. a blood sample), a population of cells, or a tissue. In particular, the term "sample" means a musculature sample or a blood sample obtained from the patient. A musculature sample may be obtained from patients who underwent bariatric surgery (e.g. laparoscopic sleevegastrectomy) for obesity, as described in EXAMPLE. A musculature sample may be collected using methods well known in the art, such as biopsy. Preferably the blood sample, according to the invention, is a plasma sample. A plasma sample may be obtained using methods well known in the art. For example, blood may be drawn from the patient following standard venipuncture procedure on tri-sodium citrate buffer. Plasma may then be obtained from the blood sample following standard procedures including but not limited to, centrifuging the blood sample at about 2500*g for about 15 minutes (room temperature), followed by pipeting of the plasma layer. Platelet-free plasma (PFP) is obtained following a second centrifugation at about 2500*g for 15 min. Analyses can be performed directly on this PFP.

[0021] As used herein, the term "PDK4" refers to pyruvate dehydrogenase kinase 4, an enzyme that regulates the activity of pyruvate dehydrogenase complex (PDC) in the mitochondria. PDC is responsible for the conversion of pyruvate into acetyl-CoA, which can then enter the tricarboxylic acid cycle or be used for fatty acid synthesis. PDK4 inhibits PDC by phosphorylating its subunits, thus reducing the oxidation of pyruvate and increasing the availability of substrate for gluconeogenesis and ketogenesis. An exemplary amino acid sequence for PDK4 is shown as SEQ ID NO:1.

[0022] SEQ ID NO : 1 >sp | Q16654 | PDK4 HUMAN [ Pyruvate dehydrogenase ( acetyl- trans ferring ) ] kinase isozyme 4 , mitochondrial 0S=Homo sapiens OX=9606 GN=PDK4 PE=1 SV=1 MKAARFVLRSAGSLNGAGLVPREVEHFSRYSPSPLSMKQLLDFGSENACERTSFAFLRQE LPVRLANILKEIDILPTQLVNTSSVQLVKSWYIQSLMDLVEFHEKSPDDQKALSDFVDTL IKVRNRHHNWPTMAQGI IEYKDACTVDPVTNQNLQYFLDRFYMNRI STRMLMNQHILI F SDSQTGNPSHIGSIDPNCDWAWQDAFECSRMLCDQYYLSSPELKLTQVNGKFPDQPIH IVYVPSHLHHMLFELFKNAMRATVEHQENQPSLTPIEVIWLGKEDLTIKI SDRGGGVPL RI IDRLFSYTYSTAPTPVMDNSRNAPLAGFGYGLPI SRLYAKYFQGDLNLYSLSGYGTDA I IYLKALSSESIEKLPVFNKSAFKHYQMSSEADDWCI PSREPKNLAKEVAM

[0023] As used herein, the term "ANGPTL4" refers to angiopoietin-like 4, a protein that regulates lipid metabolism and inflammation. ANGPTL4 inhibits lipoprotein lipase (LPL), an enzyme that hydrolyzes triglycerides in lipoproteins and releases free fatty acids for uptake by tissues. By inhibiting LPL, ANGPTL4 increases plasma triglyceride levels and reduces fatty acid utilization by muscle and adipose tissue. ANGPTL4 also modulates the inflammatory response by affecting the expression of cytokines, chemokines, and adhesion molecules. An exemplary amino acid sequence for ANGPTL4 is shown as SEQ ID NO:2.

[0024] SEQ ID NO : 2 >sp | Q9BY76 | ANGL4 HUMAN Angiopoietin-related protein 4 0S=Homo sapiens OX=9606 GN=ANGPTL4 PE=1 SV=2 MSGAPTAGAALMLCAATAVLLSAQGGPVQSKSPRFASWDEMNVLAHGLLQLGQGLREHAE RTRSQLSALERRLSACGSACQGTEGSTDLPLAPESRVDPEVLHSLQTQLKAQNSRIQQLF HKVAQQQRHLEKQHLRIQHLQSQFGLLDHKHLDHEVAKPARRKRLPEMAQPVDPAHNVSR LHRLPRDCQELFQVGERQSGLFEIQPQGSPPFLVNCKMTSDGGWTVIQRRHDGSVDFNRP WEAYKAGFGDPHGEFWLGLEKVHSITGDRNSRLAVQLRDWDGNAELLQFSVHLGGEDTAY SLQLTAPVAGQLGATTVPPSGLSVPFSTWDQDHDLRRDKNCAKSLSGGWWFGTCSHSNLN GQYFRSI PQQRQKLKKGI FWKTWRGRYYPLQATTMLIQPMAAEAAS

[0025] The expression level of the biomarker, namely PDK4 and / or ANGPTL4 can be determined by any assay well known in the art, depending on the nature and availability of the biological sample. In particular, the expression level may be determined at protein or mRNA level.

[0026] For example, if the biological sample is a tissue sample, such as a biopsy or a surgical specimen, the expression level of the biomarker may be assessed by immunohistochemistry (IHC), which is a technique that uses antibodies to detect and visualize proteins in tissue sections. IHC can provide both qualitative and quantitative information about the presence and localization of the biomarker in the tissue. IHC can also be combined with other techniques, such as in situ hybridization, to measure both protein and mRNA expression of the biomarker simultaneously. Alternatively, if the biological sample is a fluid sample, such as blood, plasma, serum, urine, saliva, cerebrospinal fluid, or synovial fluid, the expression level of the biomarker may be measured by enzyme-linked immunosorbent assay (ELISA), which is a technique that uses antibodies to capture and quantify proteins in a liquid phase. ELISA can provide sensitive and specific detection of the biomarker in the fluid sample, as well as information about its concentration and dynamics.

[0027] Another option for determining the expression level of the biomarker is to analyze the mRNA level of the biomarker in the biological sample. This can be done by various methods, such as quantitative polymerase chain reaction (qPCR), reverse transcription PCR (RT-PCR), microarray, or RNA sequencing. These methods can amplify and detect the mRNA transcripts of the biomarker in the biological sample, and provide information about their abundance and variation. qPCR and RT-PCR are based on the use of primers and probes that are complementary to the target mRNA sequence, and can produce real-time or end-point measurements of the biomarker expression. Microarray and RNA sequencing are based on the hybridization or sequencing of the mRNA molecules in the biological sample, and can produce high-throughput and comprehensive data of the biomarker expression, as well as other genes that may be involved in the disease or condition. The expression level may be expressed as absolute level or normalized level. Typically, levels are normalized by correcting the absolute level of a gene by comparing its expression to the expression of a gene that is not a relevant for determining the risk of mobility disorder, e.g., a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the actin gene ACTB, or ribosomal 18S gene. This normalization allows the comparison of the level in one sample, e.g., a patient’s sample, to another sample, or between samples from different sources.

[0028] Typically, high expression levels of PDK4 and / or ANGPTL4 indicate that the subject is at high risk of motility disorder. Conversely, low levels of PDK4 and / or ANGPTL4 indicate that the subject is at low risk of motility disorder.

[0029] As used herein, the term “high” refers to a measure that is significantly greater than normal, greater than a standard, such as a predetermined reference value or a subgroup measure, or that is relatively greater than another subgroup measure. For example, a high expression level of PDK4 and / or ANGPTL4 refers to an expression level of PDK4 and / or ANGPTL4 that is greater than a normal PDK4 and / or ANGPTL4 expression level. A normal PDK4 and / or ANGPTL4 expression level may be determined according to any method available to one skilled in the art. A high expression level of PDK4 and / or ANGPTL4 may also refer to an expression level equal to or greater than a predetermined reference value, such as a predetermined cutoff. A high expression level of PDK4 and / or ANGPTL4 may also refer to an expression level of PDK4 and / or ANGPTL4 wherein a high PDK4 and / or ANGPTL4 subgroup has relatively greater expression levels of PDK4 and / or ANGPTL4 than another subgroup. For example, without limitation, according to the present specification, two distinct patient subgroups can be created by dividing samples around a mathematically determined point, such as, without limitation, a median, thus creating a subgroup whose measure is high (i.e., higher than the median) and another subgroup whose measure is low. In some cases, a “high” expression level may comprise a range of expression levels that is “very high” and a range of expression levels that is “moderately high”, where moderately high is an expression level that is greater than normal but less than “very high”.

[0030] Thus, in some embodiments, the method of the present invention comprises i) determining the expression level of PDK4 and / or ANGPTL4 in a sample obtained from the patient, ii) comparing the expression level determined at step i) with a predetermined reference value and iii) concluding that the patient has or is at risk of having a motility disorder when the expression level determined at step i) is higher than the predetermined reference value.

[0031] Typically, the predetermined reference value is a threshold or cutoff value. Typically, a "threshold value" or "cutoff value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based on the existing experimental and / or clinical conditions, as would be recognized by a person of ordinary skill in the art. For example, retrospective measurement in properly banked historical subject samples may be used in establishing the predetermined reference value. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit / risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. For example, after determining the expression level of PDK4 and / or ANGPTL4 in a group of reference, one can use algorithmic analysis to statistically treat the levels determined in samples to be tested and thus obtain a classification standard having significance for sample classification. The full name of the ROC curve is the receiver operator characteristic curve, which is also known as the receiver operation characteristic curve. It is mainly used for clinical and biochemical diagnostic tests. The ROC curve is a comprehensive indicator that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1-specificity). It reveals the relationship between sensitivity and specificity with the image composition method. A series of different cutoff values (thresholds or critical values, boundary values between normal and abnormal diagnostic test results) are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate, and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis. On the ROC curve, the point closest to the far upper left of the coordinate diagram is a critical point with high sensitivity and specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result improves as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate. When AUC is higher than 0.9, the accuracy is high. This algorithmic method is preferably done with a computer. Existing software or systems in the art may be used to draw the ROC curve, such as MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER. SAS, CREATE-ROC.SAS, GB STAT VIO.O (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc.

[0032] In some embodiments, the predetermined reference value is the expression level of PDK4 and / or ANGPTL4 determined in a population of healthy individuals. Typically, it is concluded that the patient suffers from motility disorder when the level of PDK4 and / or ANGPTL4 is at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100 fold higher than the level determined in a population of healthy individuals.

[0033] The present invention thus provides a novel method for diagnosing and predicting motility disorders based on the expression level of PDK4 and / or ANGPTL4 in patients. This method can help physicians to identify patients who are at risk of developing or worsening motility disorders, in particular stomach dysmotility. For instance, some drugs that are commonly used to treat diabetes, such as metformin or thiazolidinediones, can induce or increase the expression of PDK4 and / or ANGPTL4, thereby potentially aggravating motility disorders. Therefore, by using the method of the present invention, physicians can monitor the expression level of PDK4 and / or ANGPTL4 in patients who are taking these drugs and adjust the dosage or switch to alternative drugs if necessary. Conversely, some drugs that can lower the expression of PDK4 and / or ANGPTL4, such as statins, fibrates, or resveratrol, may have beneficial effects on motility disorders and can be prescribed as adjuvant therapies. Thus, the method of the present invention can provide valuable information for the management of patients with or at risk of having motility disorders.

[0034] In particular, patients at risk of having a motility disorder can be treated with inhibitors of the PPARD / PDK4 / ANGPTL4 pathway.

[0035] Thus a further object of the present invention relates to a method of treating a motility disorder in patient in need thereof comprising administering to the patient a therapeutically effective amount of a inhibitor of the PPARD / PDK4 / ANGPTL4 pathway.

[0036] As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

[0037] As used herein, the term "PPARD / PDK4 / ANGPTL4 pathway" refers to a molecular signaling cascade that involves the peroxisome proliferator-activated receptor delta (PPARD), pyruvate dehydrogenase kinase 4 (PDK4), and angiopoietin-like protein 4 (ANGPTL4). This pathway plays a key role in regulating the metabolism of fatty acids and glucose in gastrointestinal muscles. Activation of PPARD by its ligands, such as fatty acids or synthetic agonists, induces the transcription of PDK4 and ANGPTL4 genes, which encode enzymes that inhibit the oxidation of glucose and promote the utilization of fatty acids as energy sources. This pathway modulates the contractility and motility of the gastrointestinal tract. Therefore, dysregulation of this pathway contributes to the development or exacerbation of motility disorders, in particular stomach dysmotility. As used herein, the term "inhibitor of the PPARD / PDK4 / ANGPTL4 pathway" refers to a compound or a combination of compounds that can reduce or block the activity or expression of one or more components of the PPARD / PDK4 / ANGPTL4 pathway. For example, such inhibitors may include antagonists or modulators of PPARD, inhibitors of PDK4 or ANGPTL4, or suppressors of PDK4 or ANGPTL4 gene transcription. Such inhibitors may be natural or synthetic, small molecules or biologies, and may act by direct or indirect mechanisms. By inhibiting the PPARD / PDK4 / ANGPTL4 pathway, these compounds can enhance the oxidation of glucose and reduce the utilization of fatty acids as energy sources in gastrointestinal muscles, thereby improving the contractility and motility of the gastrointestinal tract. Thus, these inhibitors may have therapeutic effects on motility disorders, in particular stomach dysmotility.

[0038] In some embodiments, the inhibitor of the PPARD / PDK4 / ANGPTL4 pathway may be an inhibitor of the expression of PPARD, PDK4 or ANGPTL4 genes. Such inhibitors may include, but are not limited to, small interfering RNA (siRNA), antisense oligonucleotides, microRNAs, or other molecular tools that can target the mRNA or DNA of these genes and reduce or inhibit their transcription or translation. Antisense oligonucleotides include antisense RNA molecules and antisense DNA molecules, that can bind to the mRNA or DNA of these genes and prevent their translation or increase their degradation, thus decreasing the levels and activities of PPARD, PDK4 or ANGPTL4 in gastrointestinal cells. Antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding PPARD, PDK4 or ANGPTL4 can be synthesized by conventional phosphodiester techniques or other methods known in the art (e.g., see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). Alternatively, small interfering RNAs (siRNAs) can also function as inhibitors of expression of PPARD, PDK4 or ANGPTL4 genes. The expression of these genes can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that PPARD, PDK4 or ANGPTL4 gene expression is specifically inhibited by RNA interference (RNAi).

[0039] In some embodiments, the inhibitor of the PPARD / PDK4 / ANGPTL4 pathway is small organic molecule. For the purpose of this invention, the term "small organic molecule" refers to a low molecular weight compound that contains carbon atoms and may also contain other elements, such as hydrogen, oxygen, nitrogen, sulfur, phosphorus, or halogens. Small organic molecules typically have a molecular weight of less than about 1000 Da, and may be synthesized by chemical reactions or isolated from natural sources. Small organic molecules may have various structures and functional groups, and may interact with biological targets by binding, inhibiting, activating, or modulating them. Examples of small organic molecules include, but are not limited to, benzimidazoles, pyrimidines, quinolines, quinoxalines, indoles, imidazoles, triazoles, thiazoles, oxazoles, isoxazoles, pyrazoles, furans, thiophenes, pyrroles, piperazines, piperidines, pyridines, pyrazines, morpholines, tetrahydrofurans, lactams, lactones, esters, amides, acids, alcohols, amines, ethers, ketones, aldehydes, and derivatives thereof.

[0040] In some embodiments, the inhibitor of the PPARD / PDK4 / ANGPTL4 pathway is a PPARD antagonist that can block the binding of endogenous or exogenous ligands to PPARD and prevent its activation. PPARD antagonists may have various chemical structures and modes of action, and may be identified by screening methods known in the art (e.g. Markt P, Petersen RK, Flindt EN, Kristiansen K, Kirchmair J, Spitzer G, Distinto S, Schuster D, Wolber G, Laggner C, Langer T. Discovery of novel PPAR ligands by a virtual screening approach based on pharmacophore modeling, 3D shape, and electrostatic similarity screening. J Med Chem. 2008 Oct 23;51(20):6303-17. doi: 10.1021 / jm800128k. Epub 2008 Sep 27. PMID: 18821746). Specific examples of PPARD antagonist include GSK3787 and GSK0660, which have been shown to inhibit PPARD-mediated transcriptional activity and suppress the expression of PDK4 and ANGPTL4 genes in various cell types (e.g. Shearer et al (2008) Identification and characterization of a selective peroxisome proliferator-activated receptor f / b (NR1C2) antagonist. Mol.Endocrinol. 22 523 PMID: 17975020).

[0041] According to the invention, the inhibitor of the PPARD / PDK4 / ANGPTL4 pathway is administered to the patient in a therapeutically effective amount. By a "therapeutically effective amount" is meant a sufficient amount of the active ingredient for treating or reducing the symptoms at reasonable benefit / risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination with the active ingredients; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, typically from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg / kg to about 20 mg / kg of body weight per day, especially from about 0.001 mg / kg to 7 mg / kg of body weight per day.

[0042] Typically the active ingredient of the present invention (e.g. inhibitor of the PPARD / PDK4 / ANGPTL4 pathway) is combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. The term "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. In the pharmaceutical compositions of the present invention, the active ingredients of the invention can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.

[0043] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0044] FIGURES:

[0045] Figure 1: Evaluation of the differentiation status of gastric smooth muscle in HFD- induced obese mice. Western-blot image of whole stomach extracts from adult mice fed with a HFD for 12 weeks (n = 7), and from controls (n = 7) probed with antibodies directed against specific smooth muscle proteins (Sm22, Calponinl, and gSma) and against Gapdh as loading control. Quantification of the Western-blot comparing extracts from HFD stomach extracts from controls. Data are presented as the mean ± SEM (two-tailed Mann-Whitney test; *P < 0.05; **P < 0.01). The expression of Sm22 and Calponinl was significantly lower in HFD compared to control condition.

[0046] Figure 2 : Impact of lipid treatment on human gastric smooth muscle cells. A Levels of total ceramides (left panel) and total sphingomyelins (right panel) quantified in SMCs with or without lipid treatment for an additional 3 and 7 days. All data were normalized to the quantity of protein (pg / mg of proteins). Values are the mean ±SEM of n = 6 samples. *P < 0.05 and **P < 0.01 (non-parametric Kruskall-Wallis test with Dunn’ s multiple comparison test). B Quantification of Western blot assays comparing extracts from lipid-treated SMCs to extracts from control SMCs. Data are presented as the mean ± SEM and two-tailed Mann-Whitney test was applied (*P < 0.05; ns > 0.05). CALPONIN1 expression was significantly reduced after 3 days, while SM22 was significantly lower only after 7 days of treatment (B).

[0047] Figure 3: Identification and function of the molecular mechanism induced by lipid treatment in human gastric SMC. (A) Quantification of Western blot assays comparing extracts treated for 3 days with si-NEG alone, si-7VEG+lipids, and si- / 7W- / +lipids. Data are presented as the mean ± SEM (nonparametric Kruskall-Wallis test with Dunn’s multiple comparison test: *P < 0.05, ***p < 0.001, ns > 0.05). Expression of SM22 and CALPONIN1 were decreased in lipid treatment compared to control condition and sustained with the presence of si-PDK4. (B) Quantification of Western blot assays comparing extracts treated for 3 days with si-NEG alone, si-7VEG+lipids, and si-4VG7J7 / .- / +lipids. Data are presented as the mean ± SEM (nonparametric Kruskall-Wallis test with Dunn’s multiple comparison test: *P < 0.05, **P < 0.01, ***p < 0.001, ns > 0.05). Expression of SM22 and CALPONIN1 were decreased in lipid treatment compared to control condition and sustained with the presence of si- ANGPTL4.

[0048] Figure 4: Regulation of the expression of PDK4 and ANGTPL4 mRNA expression under lipid treatment. (A) Percentage of high-level of nuclear PPARD-positive human gastric SMCs relative to the total number of nuclei (Hoechst staining) determined by immunofluorescence after treatment for 1 day with Lipid alone and with Lipid+GSK0660 (5 pM). Lipid+GSK0660 treatment reduces by 6-fold the number of nuclear PPARD-positive human gastric SMCs. (B) RT-qPCR of PDK4 (left panel) and ANGPTL4 (right panel) relative mRNA expression in human gastric SMC cultures treated for 3 days with GSK0660 (PPARD antagonist, 5 pM), with lipid, and with lipid+GSK0660 compared to untreated SMC (Control). Data were normalized to house-keeping HMBS expression. Values are the mean ± SEM of n=6 samples. ****p < 0.0001, ns > 0.05 (One way ANOVA, multiple comparison test). PDK4 and ANGPTL4 stimulation induced by lipid treatment are diminished after 3 days of GSK0660 treatment, close to the levels observed in the untreated condition (Control).

[0049] Figure 5: Evaluation of the differentiation status of human gastric smooth muscle in patients with obesity. (A) Summary of the histological evaluation of stomach sections from adult patients with obesity (n = 15) and adult controls (n = 4) using Hematoxylin and Eosin staining, along with immunohistochemical staining for the smooth muscle marker CALPONIN1. After staining, gastric smooth muscle was evaluated and scored independently for their organization into normal, and affected regions by three pathologists and consensus was reported. (B) Quantification of Western-blot assays comparing extracts from adult patients with obesity (n = 15) to extracts from controls (n = 4). Data are presented as the mean ±SEM (two- tailed Mann-Whitney test; *P < 0.05). Notably, the expression of SM22 and CALPONIN 1 were found significantly lower in patients with obesity compared to controls.

[0050] Figure 6: Evaluation of the expression of PDK4, ANGPTL4 and LIX1 expression and their correlation in patients with obesity. (A) RT-qPCR of PDK4 relative mRNA expression of gastric smooth muscle fiber extracts from adult patients with obesity (n=15), and from controls (n=4). Data were normalized to house-keeping HMBS expression. Values are the mean ± SEM and two-tailed Mann-Whitney test was applied (**P < 0.01). PDK4 is statically found 13-fold induced in patients with obesity. (B) RT-qPCR oiANGPTL4 relative mRNA expression of gastric smooth muscle fiber extracts from adult patients with obesity (n=15), and from controls (n=4). Data were normalized to house-keeping HMBS expression. Values are the mean ± SEM and two-tailed Mann-Whitney test was applied (ns > 0.05). ANGPTL4 is found 3.5-fold induced in patients with obesity. (C, D) The correlation between PDK4 and ANGPTL4 expression or SM22 expression were calculated by Two-tail Pearson’s correlation test. P and R values are indicated on each graph. Positive correlation was identified between PDK4 and ANGPTL4 (C), whereas negative correlation was found between PDK4 and SM22 (D). (E) RT- qPCR of LIX1 relative mRNA expression of gastric smooth muscle fiber extracts from adult patients with obesity (n=15), and from controls (n=4). Data were normalized to house-keeping HMBS expression. Values are the mean ± SEM and two-tailed Mann-Whitney test was applied (***P < 0.001). LIX1 is statically found to be 22-fold upregulated in patients with obesity. (F- H) The correlation between LIX1 and SM22 expression, or PDK4 and ANGPTL4 expression were calculated by Two-tail Pearson’s correlation test. P and R values are indicated on each graph. Negative correlation was identified between LIX1 and SM22 expression (F), where positive correlations are found between LIX1 and PDK4 (G) or ANGPTL4 expression (H).

[0051] Figure 7: Schematic model of the pathway leading to alterations in gastric smooth muscle in patients with obesity. Upon lipid exposure or specific agonist (GW501516), the ligand- activated transcription factor PPARD becomes active. These regulations correlate with the nuclear translocation shuttling of the PPARD protein, supporting a potential transcriptional control at the peroxisome proliferator response elements (PPREs) of PDK4 and ANGPTL4 promoter, which still needs to be validated in our model. Upregulation of PDK4 and ANGPTL4 is crucial for initiating the dedifferentiation process of gastric SMCs observed in patients with obesity.

[0052] EXAMPLE:

[0053] Methods:

[0054] Patients

[0055] The study involved collecting gastric wall samples from two patient groups. The first group included 15 patients with obesity who underwent sleeve gastrectomy between September and December 2018. The second group comprised 4 non-obese control patients with a body mass index of less than 30 kg / m2 who had surgeries for gastric or esophageal epithelial tumors in January 2019. Participants were included regardless of their weight or diabetic status, and their biological data were collected. Samples were collected in accordance with the ethical guidelines of Montpellier University Hospital (France), with informed consent obtained from each patient, permitting the use of their tissue samples for research purposes. Micro-dissected smooth muscle fibers from the body part of the stomach were analyzed using Western blotting and quantitative RT-PCR. Additionally, biological data were collected for each patient group.

[0056] High Fat Diet mice treatment

[0057] Male C57BL / 6J mice (10-11 weeks old) from Janvier Laboratories (Saint-Berthevin Cedex, France) were individually housed under controlled conditions with a 12-hour light-dark cycle and had unrestricted access to food and water. The study adhered to the European Parliament Directive 2010 / 63 / EU and was approved by the local ethics committee of Marseille (Approval Number: 2020050612125728). Mice were randomly divided into two groups (n=7 per group): one group received a normal chow diet (Control) and the other a High Fat Diet (HFD). After 12 weeks of diet, mice were assessed for changes in metabolism, histology, and biochemistry.

[0058] Human gastric smooth muscle cell and treatment

[0059] Human gastric SMCs from Innoprot Innovative (Spain) were cultured on collagen Lcoated dishes (Coming) in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine serum and 1% penicillin / streptomycin

[0027] , Differentiation was induced over 14 days. Pharmacological treatments began after differentiation. For lipid treatments, SMCs were exposed to a 2% lipid mixture with BSA-complexed long-chain fatty acids, 30 pM palmitic acid, and 30 pM oleic acid, or a 60 pM BSA control for 3 or 7 days. In PPARD antagonist experiments, SMCs were serum-starved for 24 hours and treated with 5 pM GSK0660 or 0.1% DMSO for 24 hours, followed by exposure to the lipid mixture with additional GSK0660 every 24 hours for 72 hours. For PPARD agonist experiments, SMCs were treated with 1 pM GW501516 or 0.1% DMSO for 72 hours. To inhibit lipid-induced PDK4 and ANGPTL4 expression, SMCs were treated with WRAP5 -siRNA nanoparticles targeting PDK4, ANGPTL4, or a control siRNA for 2 hours, then cultured in a lipid mixture for 3 days. Analysis included immunofluorescence, Western blotting, quantitative RT-PCR, and assessments of cell viability and toxicity. Gastric SMCs were routinely screened for mycoplasm contamination using the MycoAlert® Detection Kit (Lonza).

[0060] Transcriptomic sequencing and lipidomic analysis

[0061] The gene expression profile of human gastric SMC cultures treated with fatty acids complexed with BSA, along with their respective controls, was assessed using high-throughput RNA sequencing. Differentially expressed genes were identified using the DESeq2 statistical method (MGX platform, Biocampus, Montpellier, France). The lipid composition of the treated SMC cultures was analyzed using Liquid chromatography / tandem mass spectrometry (LC-MS / MS) measurements (Metatoul platform, Toulouse, France) as previously described

[0032] ,

[0062] Statistical analysis

[0063] Data are shown as means ± standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism 9.0 software (GraphPad, San Diego, CA) using the two-tailed Mann-Whitney test. For multiple comparisons within groups, the Kruskal-Wallis test followed by Dunn’s multiple comparisons test were employed. Details of the statistical tests used are provided in the legend of each figure. A p-value of less than 0.05 was considered statistically significant.

[0064] Results:

[0065] Obesity is associated with gastric musculature alterations in humans and mice

[0066] To investigate potential changes in gastric musculature associated with obesity, we first analyzed 15 samples from patients who underwent laparoscopic sleeve gastrectomy for obesity and 4 samples from non-obese patients (controls) associated with gastric or esophageal carcinoma. Histopathology and immunohistochemistry focused on smooth muscle (CALPONIN1) marker. While some patients with obesity showed discrete changes in the smooth muscle layer organization, more than 66% of patients with obesity showed muscle alterations (data not shown). No alterations were found in the control subjects. Next, we evaluated the differentiation status of gastric smooth muscle cells (SMCs) using immunodetection on whole gastric musculature and western blotting from dissected gastric smooth muscle fibers. Western blot analysis revealed significant decrease in the expression levels of differentiated markers such as SM22 and CALPONIN1 in patients with obesity compared to controls. In contrast, the expression level of ySMA, a marker that defines SMC determination and identity, remained unchanged indicating a specific deregulation of the differentiation status of the SMCs (data not shown). Immunochemistry analysis confirmed the reduced level of CALPONIN1 in the musculature of patients with obesity compared to controls (data not shown).

[0067] We then analyzed the gastric musculature in a mouse model of early induced obesity. Male adult mice were subjected to a High fat diet (HFD) with 60% fat (230-HFD) for 12 weeks, a period known to induce various obesity-related metabolic changes

[0033] , This dietary regimen effectively induced obesity, as evidenced by the appearance of multiple metabolic phenotypes commonly associated with the condition (data not shown). Using immunofluorescence approaches, we first evaluated the differentiation status of gastric smooth muscle cells (SMCs) using specific SMC markers, respectively gSma, a marker defining SMC determination and identity and Sm22, a marker of SMC differentiation status. We observed a reduced level of Sm22 in the gastric musculature of HFD mice compared to controls, whereas the expression level of gSma remained unchanged (data not shown). To verify the presence and integrity of enteric neurons in the stomachs of HFD-treated mice, we used enteric neuronal marker (Tuj 1) marker and observed that Tuj 1 signal across experimental groups is preserved supporting that the enteric neuronal network remained largely intact (data not shown). Western blot analysis revealed a significant reduction in Sm22 and Calponinl levels in gastric extracts prepared from obese mice compared to controls (Figure 1). ySMA levels remained constant (Figure 1) indicating a specific deregulation of the differentiation status of the SMCs, similar to what we observed in human samples. These findings suggest a link between obesity and alterations in the differentiation status of gastric smooth muscle.

[0068] Lipid treatment altered gastric smooth muscle cell differentiation

[0069] Obesity has been demonstrated to perturb both intestinal epithelium homeostasis and enteric nervous system composition [34,35], These could contribute to the alteration of gastric smooth muscle observed above through epithelial-mesenchymal or neuro-mesenchymal interactions [36,37], To explore whether dietary HFD components can directly contribute to the gastric smooth muscle phenotype observed in HFD-induced obesity mice, we set-up an in vitro culture model of differentiated human gastric SMCs. In vitro studies on SMCs are challenging due to the spontaneous and uncontrolled phenotypic modulation of SMCs toward synthetic and undifferentiated states [22-25,27,38], Research on uterine SMCs indicates that low-density culture reduces contractile marker expression, while confluence induces loss of proliferative capacity and upregulation of differentiation markers

[0039] , Culturing ySMA-positive human gastric SMCs to confluence over a period of 14 days induced the expression of SM22 and CALPONIN1, hereafter referred to as differentiated SMCs (data not shown). We then exposed differentiated SMCs to lipid constituents of the HFD including long-chain fatty acids supplemented with palmitate acid and oleate acid, as previously described

[0034] , Treating differentiated SMCs with the lipid mixture resulted to its rapid cellular internalization, as evidenced by Bodipy detection after 6 hours observed through confocal microscopy (data not shown). This was followed by sustained intracellular localization in lipid droplets, as observed through transmission electron microscopy after 7 days of lipid treatment (data not shown). We verified that lipid treatment did not affect the viability of SMCs after 3 and 7 days of treatment (data not shown). Analysis of intramembranous lipid composition using liquid chromatography / tandem mass spectrometry lipidomic approach revealed increases in the fatty acid content of the SMC membrane at 3 and 7 days of treatment (data not shown). This was associated by a specific decrease of total ceramides with no change in total sphingomyelins, suggesting a pre-metabolic condition (Figure 2A). We next evaluated the impact of lipid treatment on SMC homeostasis. Importantly, we found that lipid treatment was associated with a decrease in the levels of both SM22 and CALPONIN1 proteins, while ySMA expression remained unchanged (Figure 2B). These data demonstrate a direct role of lipids on SMC dedifferentiation and plasticity.

[0070] PDK4 and ANGPTL4 are essential in lipid-induced dedifferentiation of human gastric SMCs

[0071] To gain a deeper understanding of how lipid treatment mediates these effects, we performed RNA sequencing analysis on human gastric SMCs at 3 and 7 days of lipid treatment. Using a significance cutoff of p < 0.01, we identified 139 up-regulated and 88 down-regulated genes compared to unstimulated SMCs at both time points (data not shown). Notably, PDK4 and ANGPTL4 were found strongly upregulated (data not shown). Stimulation of PDK4 and ANGPTL4 transcript level at 3 and 7 days of treatment with the lipid mixture was confirmed by RTqPCR (data not shown). These upregulations are specific to the lipid concentration used (data not shown). Pyruvate dehydrogenase kinase family proteins (PDKs 1-4) inhibit glycolysis-dependent oxidative phosphorylation (OXPHOS) by inactivating pyruvate dehydrogenase complex, while angiopoi etin-like proteins (ANGPTLs 1-8) are involved in various physiological and pathological functions related to tissue repair and homeostasis [40,41], Among the PDK and ANGPTL family members, only PDK4 and ANGPTL4 were upregulated in response to lipid treatment (data not shown). Moreover, we found that PDK4 and ANGPTL4 mRNAs are rapidly induced by lipids treatment in a dose dependentmanner (data not shown).

[0072] To investigate the role of PDK4 and / or ANGPTL4 upregulation in the dedifferentiation of human gastric SMCs, we employed an efficient method of cellular delivery of siRNA targeting these genes without using lipid-based siRNA transfection systems. For this purpose, we utilized a 16-mer tryptophan- and arginine-rich amphipathic peptide (WRAP)

[0042] , which forms nanoparticles with siRNA molecules, averaging 100 nm in size and exhibiting no cytotoxicity on human gastric SMCs (data not shown). We demonstrated that Cy5-labeled WRAP5-siRNA nanoparticles were efficiently internalized by nearly all human gastric SMCs within 2 hours (data not shown). Targeting PDK4 or ANGPTL4 with siRNAs successfully blocked the upregulation induced by lipid treatment (data not shown), leading to SM22, CALPONIN1, and ySMA expression levels similar to those of unstimulated SMCs (Figure 3A-B).

[0073] To further characterize the impact of lipids on SMC dedifferentiation, we extended the duration of lipid exposure to 14 days. Lipid treatment led to a consistent reduction in CALPONIN1 and SM22 expression at both 7 and 14 days, indicating persistent and stable dedifferentiation over time (data not shown). While ANGPTL4 induction remained comparable between time points, PDK4 showed a marked increase over time, suggesting a differential temporal regulation of these two genes by lipids (data not shown). We next assessed the capacity for phenotypic recovery following lipid removal. After 7 days of lipid treatment, human gastric SMCs were cultured for an additional 7 days in standard medium. PDK4 and ANGPTL4 transcript levels returned to baseline upon lipid withdrawal, supporting a reversible induction response to lipid exposure (data not shown). Under these conditions, SM22 expression also returned to baseline, whereas CALPONIN1 expression remained repressed, indicating a partial restoration of the differentiated phenotype (data not shown). As previously observed, ySMA expression remained unchanged across conditions.

[0074] Overall, our findings highlight the crucial role of PDK4 and ANGPTL4 as key relays in driving the dedifferentiation of human gastric SMCs in the presence of lipids.

[0075] PPARD regulates the induction of PDK4 and ANGPTL4 in lipid-treated gastric SMCs

[0076] We sought to gain further insight into the mechanisms by which lipid mixture treatment induces the expression of PDK4 and ANGPTL4. We evaluated whether this effect arises from mRNA stabilization or transcriptional activation. Given our previous findings that RNA-binding proteins can stabilize specific mRNAs to promote SMC dedifferentiation

[0070] , we first investigated potential changes in mRNA stability. Actinomycin D chase experiments revealed that PDK4 and ANGPTL4 transcripts were similarly degraded over time in both control and lipid-treated SMCs. No significant differences in mRNA half-life were observed, indicating that lipid-induced upregulation does not result from increased transcript stability, but rather supports a transcriptional mechanism of regulation (Data not shown). Peroxisome proliferator- activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear hormone receptor superfamily, which include three members PPARa, PPARy, and PPARp / 5 (also name PPARD)

[0043] , These receptors can be activated by various ligands, including fatty acids

[0044] , Upon ligand binding, PPARs form heterodimers with retinoid X receptors (RXRs), and this PPAR / RXR complex is crucial for binding to specific DNA regions known as PPAR response elements (PPREs) in the gene promoter region

[0045] , PPARD has been shown to directly bind to the minimal promoter of PDK4, thereby upregulating its expression

[0046] , Bioinformatic analysis of PDK4 and ANGPTL4 promoters using the ConTraV3 browser highlighted the presence of PPAR-RXRa binding sites in 5’ region. These sites are highly conserved across vertebrate species for PDK4 and conserved exclusively in primates for ANGPTL4 (data not shown). Notably, PPARD is the most abundantly expressed member of the PPAR family in human gastric SMCs (data not shown), and its expression is rapidly and transiently induced by lipid treatment in association with PDK4 and ANGPTL4 mRNA upregulation (data not shown). Transcriptional PPARD activity may be linked to its nuclear localization

[0044] , we assessed the localization of PPARD in human gastric SMCs under lipid treatment and found that lipid increases the number of cells with PPARD-positive nuclei compared to controls (data not shown). The activation of PPARD activity using GW501516, a selective PPARD agonist, resulted in a significant increase of PDK4 and ANGPTL4 mRNA levels (data not shown). Notably, GW501516 treatment was statistically more potent than lipid treatment in inducing PDK4 and ANGPTL4 expression (data not shown). Furthermore, we evaluated the impact of PPARD activation on SMC homeostasis. After 1 day, GW501516 treatment induces a decrease in the levels of both SM22 and CALPONIN1 proteins, while ySMA expression remained unchanged (data not shown). Additionally, sustained PPARD activation through GW501516 treatment led to a decrease in the expression levels of SM22, CALPONIN1, and also ySMA (data not shown) without change in PPARD level expression (data not shown). We next combined lipid treatment with GSK0660, a specific and potent PPARD antagonist, and found that GSK0660 treatment inhibits PPARD nuclear localization in lipid condition (Figure 4 A) and conducts to a partial restoration of the differentiated phenotype, with a significant increase in SM22 expression, while CALPONIN1 expression remained repressed, indicating incomplete reactivation of the mature SMC program (Data not shown). To evaluate the importance of PPARD in regulating the upregulation of PDK4 and ANGPTL4 mRNA expression under lipid stimulation, we combined lipid treatment with GSK0660, and observed an inhibition of the aforementioned stimulation (Figure 4B). These findings demonstrate that lipid treatment triggers the activation of PPARD, resulting in the upregulation of PDK4 and ANGPTL4 mRNA and consequently the induction of human gastric SMC dedifferentiation.

[0077] PDK4 and ANGPTL4 expression correlates to gastric smooth muscle dedifferentiation and the acquisition of immature features in patients with obesity

[0078] To investigate potential changes in gastric musculature in patients with obesity, we first analyzed with histopathology and immunohistochemistry approaches 15 samples from patients who underwent laparoscopic sleeve gastrectomy for obesity, as well as 4 samples from lean patients associated with gastric or esophageal carcinoma (controls) (data not shown). We found that 66% of patients with obesity showed changes in the organization of the smooth muscle layers, while no alterations were found in the control subjects (Figure 5A). Next, we evaluated the differentiation status of gastric SMCs using CALP0NIN1 and SM22 marker antibodies. Western blot analysis of extracts from dissected gastric smooth muscle fibers confirms a significant decrease in the expression levels of differentiated markers, such as SM22 and CALP0NIN1 in patients with obesity compared to controls (Figure 5B). As previously observed in mice and in SMC culture, the expression level of gSMA, a marker defining SMC determination and identity, remained unchanged indicating a specific deregulation of the differentiation status of the SMCs (Figure 5B).

[0079] We then assessed the relevance of the signaling pathway we identified using SMC culture model in patients with obesity, through the evaluation of their expressions in their gastric smooth muscle fibers (Figure 6). Our analysis revealed that PDK4 mRNA was significantly upregulated in patients with obesity compared to controls (Figure 6A), while ANGPTL4 mRNA exhibited a slight increase compared to controls (Figure 6B). Interestingly, there is a significant positive correlation between ANGPTL4 and PDK4 mRNA expressions in patients with obesity, indicating that their expressions are related (Figure 6C). Furthermore, the level of PDK4 mRNA was inversely correlated with SM22 protein expression (Figure 6D). Given that alterations in smooth muscle differentiation are often associated with mesenchymal immaturity, we investigated the expression of LIX1, a specific marker and regulator of stomach mesenchymal progenitors [26,27,47], Our analysis revealed thatLIXl mRNA was significantly upregulated in patients with obesity compared to controls (Figure 6E). Additionally, in patients with obesity, the level of LIX1 mRNA showed an inverse correlation with SM22 protein expression (Figure 6F). Importantly, LIX1 mRNA expression showed significant positive correlations with both PDK4 and ANGPTL4 mRNA expression (Figure 6G,H). These findings revealed an association between the expressions of PDK4 and ANGPTL4 and the immaturity of gastric smooth muscle in patients with obesity (Figure 7).

[0080] Discussion:

[0081] GI complications are often encountered in patients with obesity

[0014] , The GI system plays a pivotal role in regulating food intake and the development of obesity, making it a prime target for interventions. However, whether the GI tract's involvement in obesity is causative or consequential remains uncertain. Additionally, multiple and contradictory functional GI alterations are described in patients with obesity [7,14], These discrepancies likely arise from anatomical differences along the rostro-caudal axis (stomach, small intestine, colon) and their specific functions, as well as from varying approaches used for patient functional evaluation. In this study, we focused on the stomach and specifically on the differentiation status of the smooth muscle, the effector of the contraction-relaxation process. Using human stomach samples from patients with obesity and mouse stomachs from HFD-induced obesity, we identified impaired smooth muscle differentiation status in the stomach in both cases. We found that the global level of GI smooth muscle actin (ySMA)

[0048] was unchanged, while the expression of regulators of contraction (CALPONIN1 and SM22 proteins) decreased. These data suggest that gastric SMCs maintain their smooth muscle identity, but exhibit impaired contractility function. Furthermore, while LIX1 expression is typically confined to mesenchymal progenitors during the fetal period [26,27], it is elevated in the stomach smooth muscle of patients with obesity. This is indicative of a dedifferentiation process [27,38], a mechanism observed in pediatric and adult functional dysmotility syndromes such as primary visceral myopathy, inflammatory bowel disease, and achalasia [22-24,49,50], These findings are consistent with reports of accelerated gastric emptying in patients with obesity [6,11-14], The stomach is a complex organ comprising epithelial cells, enteric neurons, glial cells, and mesenchymal -derived cells such as SMCs and ICCs [8,16,51], HFD-induced obesity has been shown to prevent age-associated loss in specific populations of enteric neurons, leading to accelerated gastric emptying

[0035] , In the intestine, HFD-induced obesity increases the numbers and function of Lgr5+ intestinal epithelial stem cells in mammals

[0034] , Recent studies have investigated the immediate response of the intestinal epithelium to HFD, revealing metabolic changes within days of HFD exposure, leading to rapid intestinal epithelial adaptation [52,53], As it is well-known that the development and adult homeostasis of the GI tract require epithelial-mesenchymal interactions [8,36,54,55], we could speculate that gastric smooth muscle dedifferentiation could be either a direct impact of diet on gastric SMC or a consequence of changes in the gastric epithelium. To explore this question, we treated differentiated human gastric SMCs with a lipid mixture complemented with oleate and palmitate acids as previously used

[0034] , After lipid treatment, we found that gastric SMCs quickly internalized lipid droplets without impacting cell viability. This was associated with decreased expression levels of both SM22 and CALPONIN1 proteins, while ySMA expression remained unchanged, as previously observed in patients with obesity and HFD-induced obesity mice. This phenocopy supports the idea that lipid overload in patients with obesity likely triggers SMC dedifferentiation mechanisms, ultimately responsible for accelerated gastric emptying [6,11-14],

[0082] Using the above dedicated myologic model, we investigated the potential trigger mechanism responsible for the dedifferentiation of gastric SMCs and identified that lipid treatment rapidly induced the expression of PDK4 and ANGPTL4. PDK4, a key regulator of the pyruvate dehydrogenase complex, plays a significant role in obesity-related insulin resistance and metabolic dysfunction

[0056] , Its activity is increased in obesity, leading to enhanced formation of mitochondria-associated endoplasmic reticulum membranes and suppression of insulin signaling

[0056] , ANGPTL4 is crucial in lipid metabolism, particularly in the regulation of lipoprotein lipase activity in various tissues

[0057] , Given the roles of PDK4 and ANGPTL4 proteins in lipid metabolism — PDK4 influences fatty acid oxidation, while ANGPTL4 inhibits lipoprotein lipase activity — we investigated their involvement in gastric SMC dedifferentiation. Using siRNA-peptide-based nanoparticles to specifically inhibit the PDK4 or ANGPTL4 stimulation induced by lipid treatment, we highlighted the essential roles of both PDK4 and ANGPTL4 in lipid-induced SMC dedifferentiation, positioning these two actors in a new role during gastric SMC homeostasis and plasticity.

[0083] To explore the potential interplay between PDK4 and ANGPTL4 expression regulation, we used informatic promoter evaluation, and found the presence of PPAR-RXR binding sites in the promoters of PDK4 and ANGPTL4 genes. Previous studies have shown that PPARD is expressed during the development of the vertebrate GI mesenchyme

[0058] , and we found that PPARD is the most highly expressed member of the PPAR family in human gastric SMCs. Using specific PPARD agonist and antagonist treatments, we demonstrated that PPARD activation is sufficient to induce PDK4 and ANGPTL4 mRNA upregulation and is necessary for their induction following lipid treatment. These requirements are associated with the nuclear shuttling of PPARD protein, supporting direct regulation at the promoter level. These findings are consistent with studies in different cell types (skeletal muscle and adipocytes) suggesting that PPARD might regulate PDK4 and / or ANGPTL4 [46,59-61], Furthermore, our findings demonstrate that targeted activation of PPARD leads to a rapid downregulation of SM22 and CALPONIN1 expression, mirroring the phenotypic alterations observed following lipid treatment. Altogether, these data position the PPARD / PDK4 / ANGPTL4 pathway in a new role during gastric SMC homeostasis and plasticity.

[0084] PDK4 is predominantly expressed in skeletal muscle and heart, with significantly increased expression in the skeletal muscle of insulin-resistant patients

[0062] , Additionally, promoter methylation of PDK4 is decreased in the skeletal muscle of patients with obesity, restored to non-obese levels after Roux-en-Y gastric bypass-induced weight loss, and inversely correlated with PDK4 mRNA expression

[0063] , ANGPTL4 is a fasting-induced factor primarily expressed in the liver, adipose tissue, and ischemic tissues [41,64,65], Humans deficient in ANGPTL4 (pE40K variant) have lower triglyceride levels, higher high-density lipoprotein cholesterol levels

[0066] , and a lower risk of coronary artery disease

[0067] , Moreover, patients with the pE40K ANGPTL4 variant exhibit lower fasting glucose and greater insulin sensitivity, along with reduced risk of developing type 2 diabetes

[0068] , Although several studies have shown a correlation between serum levels of ANGPTL4 and body mass index, potentiated by the presence of type 2 diabetes

[0069] , the source and function of ANGPTL4 expression remain debated. Since PDK4 and ANGPTL4 expression had not been evaluated in human gastric smooth muscle, we assessed their mRNA expression in gastric smooth muscle fibers from patients with obesity and identified a statistically significant increase in PDK4 expression, whereas ANGPTL4 expression showed a slight difference compared to controls. When correlating PDK4 and ANGPTL4 levels with the status of smooth muscle using the expression of the digestive mesenchymal progenitor marker LIX1 [26,27], we found significant positive correlations between LIX1 and PDK4 or ANGPTL4 expression. These findings underscore the importance of the PDK4 / ANGPTL4 pathway in dysfunctional gastric smooth muscle in patients with obesity and suggest a new mechanism underlying GI alterations in obesity.

[0085] In conclusion, this study reveals a novel mechanistic link between obesity and gastric smooth muscle dysfunction, implicating the activation of the PPARD / PDK4 / ANGPTL4 pathway. The correlation of PDK4 and ANGPTL4 expression with markers of mesenchymal immaturity and smooth muscle dedifferentiation underscores their potential as biomarkers and targets for therapeutic intervention in obesity-related gastric dysmotility. These insights open up promising perspectives for the development of targeted therapeutic strategies to mitigate the GI disorders associated with obesity.

[0086] REFERENCES:

[0087] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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Claims

35 -CLAIMS:

1. A method of determining whether a patient suffering from obesity is at risk of having a motility disorder comprising the step consisting of determining the level of PDK4 and / or ANGPTL4 in a sample obtained from the patient wherein said level correlates with the risk of having a motility disorder.

2. The method according to claim 1 for determining whether the patient is at risk of having a stomach dysmotility.

3. The method according to claim 1 for determining whether the patient who underwent a bariatric surgery is at risk of having a stomach dysmotility.

4. The method according to any one of claims 1 to 3 wherein the sample is a musculature sample or a blood sample.

5. The method according to any one of claims 1 to 4 wherein high expression levels of PDK4 and / or ANGPTL4 indicate that the subject is at high risk of motility disorder whereas low levels of PDK4 and / or ANGPTL4 indicate that the subject is at low risk of motility disorder.

6. The method according to claim 5 wherein it is concluded that the patient is at risk of having a motility disorder when the level of PDK4 and / or ANGPTL4 is higher than the level determined in a population of healthy individuals.

7. A method of treating a motility disorder in patient in need thereof comprising administering to the patient a therapeutically effective amount of an inhibitor of the PPARD / PDK4 / ANGPTL4 pathway.

8. The method according to claim 7 wherein the patient was considered at risk of having a motility disorder by carrying-out the method according to any one of claims 1 to 6.

9. The method according to claim 7 or 8 wherein the inhibitor of the PPARD / PDK4 / ANGPTL4 pathway may be an inhibitor of the expression of PPARD, PDK4 or ANGPTL4 genes.

10. The method according to claim 8 or 7 wherein the inhibitor of the PPARD / PDK4 / ANGPTL4 pathway is a PPARD antagonist that can block the binding of endogenous or exogenous ligands to PPARD and prevent its activation.

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