Patent Application
20250082781
This disclosure concerns methods of reducing blood glucose and treating type 2 diabetes (T2D) in a subject by administering an effective amount of an agent that increases expression or activity of phosphodiesterase 4D isoform 3 (PDE4D3) in adipocytes of the subject.
The electronic sequence listing, submitted herewith as an XML file named 7158-107397-02 SL.xml (59,387 bytes), created on Dec. 27, 2022, is herein incorporated by reference in its entirety.
Chronic hyperglycemia and dyslipidemia are hallmarks of Type 2 diabetes mellitus (T2DM) attributed to the failure of insulin to appropriately suppress hepatic glucose production and adipose lipolysis. Moreover, unregulated lipolysis leads to the aberrant accumulation of free fatty acids (FFAs) in peripheral metabolic tissues including liver, muscle, and pancreatic islets, further exacerbating disease severity (Saponaro et al., 2015; Sears and Perry, 2015). Physiologically, adipose lipolysis is regulated, in part, by opposing hormonal stimuli that control cyclic adenosine monophosphate (cAMP) levels and protein kinase A (PKA) activity (Bartness et al., 2014; Duncan et al., 2007). Pro-lipolytic hormones (e.g., glucagon, growth hormone, thyroid hormone, cortisol, catecholamines) elevate cellular cAMP levels to drive PKA phosphorylation of key lipolytic proteins including perilipin and hormone-sensitive lipase (HSL). Conversely, insulin remains the only known anti-lipolytic hormone, acting via phosphodiesterase 3B (PDE3B) to suppress cAMP levels and inhibit PKA activity (Kitamura et al., 1999; Strålfors and Honnor, 1989; Young et al., 2006). Phosphodiesterases (PDEs) catalyze the conversion of cAMP to AMP. Eleven PDE families (PDE1-PDE11), each encompassing multiple isoforms, have been described (Azevedo et al., 2014). In adipose tissue in particular, different PDE4 isoforms have been implicated in the regulation of the cAMP/PKA pathway, however their contributions to lipolysis are not known (Baeza-Raja et al., 2016; Grønning et al., 2006; Zhang et al., 2009). Half of the PDE activity in adipocytes is attributed to PDE4A-D, where the isoform-specific N-terminal domains regulate protein-protein interactions and subcellular localization (Houslay and Adams, 2003; Young et al., 2006). Consistent with this, PDE4 inhibitors enhance lipolysis, particularly when PDE3 activity is inhibited (Dipilato et al., 2015; Grønning et al., 2006; Kraynik et al., 2013; Snyder et al., 2005). Mice deficient in PDE4A, PDE4B and PDE4D genes have been generated (Jin and Conti, 2002; Jin et al., 2005; Jin et al., 1999). Loss of PDE4A and PDE4B in adipocytes led to increased cAMP levels without affecting lipolysis (Grønning et al., 2006; Zhang et al., 2009). In contrast, adipocyte PDE4D expression is induced by insulin and synthetic catecholamines, and lower PDE4D levels are associated with increased β-adrenergic signaling, implicating a potential role in lipolysis (Jang et al., 2020; Oknianska et al., 2007).
Fibroblast growth factor 1 (FGF1) has an established role in adaptive adipose remodeling (Jonker et al., 2012; Wang et al., 2020). Mice lacking FGF1 develop a more aggressive diabetic phenotype in response to a dietary challenge (high fat diet, HFD) that is, in part, attributed to a failure to appropriately remodel adipose tissue. FGF1 expression in adipose tissue is controlled by peroxisome proliferator-activated receptor gamma (PPARγ) and is robustly induced in the fed state and upon HFD feeding (Choi et al., 2016; Jonker et al., 2012). In addition, peripheral delivery of FGF1 rapidly lowers blood glucose levels in diabetic mouse models in an adipose FGF receptor (FGFR) 1-dependent manner (Suh et al., 2014).
It is disclosed herein that FGF1 suppresses adipose lipolysis and the anti-lipolytic activities of FGF1 are required for acute glucose lowering. Further disclosed is the discovery that these FGF1 activities are mediated by the activation of phosphodiesterase 4D isoform 3 (PDE4D3) in adipose tissue. In view of these findings, the present disclosure provides methods of lowering blood glucose and/or treating Type 2 diabetes in a subject by increasing expression or activity of PDE4D3.
Provided herein are methods of reducing blood glucose in a subject and/or treating type 2 diabetes in a subject. In some aspects, the method includes administering to the subject a therapeutically effective amount of an agent that increases expression or activity of PDE4D3 in adipocytes of the subject.
In some examples, the agent that increases expression or activity of PDE4D3 includes a nucleic acid molecule encoding a PDE4D3 protein, such as a vector that includes the nucleic acid molecule operably linked to an adipocyte-specific promoter. In other examples, the agent is a nucleic acid molecule encoding the PDE4D3 protein that is introduced into adipocytes using a gene editing method.
In some examples, the agent that increases expression or activity of PDE4D3 is a small molecule activator of PDE4D3, such as an N-substituted-2-(3-aryl-1H-1,2,4-triazol-1-yl) acetamide (e.g., MR-L2). In specific examples, the small molecule activator of PDE4D3 is conjugated to an antibody that specifically binds fibroblast growth factor receptor 1b (FGFR1b), or is conjugated to an incretin, such as gastric inhibitory peptide (GIP) or glucagon-like peptide-1 (GLP-1).
In some aspects, the method further includes administering to the subject a therapeutically effective amount of a mature fibroblast growth factor 1 (FGF1) protein or a modified mature FGF1 protein, such as a modified mature FGF1 protein that has reduced mitogenicity and/or increased stability compared to native FGF1 protein, and/or selectively binds FGFR1b. In some examples, the method further includes administering a therapeutically effective amount of an additional therapeutic agent, such as an anti-diabetic agent.
Also provided are agents that can be used in such methods, such as a nucleic acid molecule encoding a PDE4D3 protein operably linked to an adipocyte-specific promoter, and vectors (such as plasmids or viral vectors) that include such. In one example, the agent is a small molecule activator of PDE4D3 conjugated to an antibody that specifically binds fibroblast growth factor receptor 1b (FGFR1b). In one example, the agent is a small molecule activator of PDE4D3 conjugated to an incretin, such as GIP or GLP-1. Compositions that include such agents are also provided, for example which may further include a pharmaceutically acceptable carrier such as water, saline, or a buffer.
The foregoing and other objects and features of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.
The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and single letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. In the accompanying sequence listing:
SEQ ID NOs: 33-35 are amino acid sequences of phosphorylation sites in mouse, rat, and human PDE4D3 (see
SEQ ID NO: 36 is the amino acid sequence of the QRRES motif.
SEQ ID NO: 37 is the amino acid sequence of the FRRHS motif.
The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. The singular forms “a,” “an,” and “the” refer to one or more than one, unless the context clearly dictates otherwise. For example, the term “comprising a cell” includes single or plural cells and is considered equivalent to the phrase “comprising at least one cell.” The term “or” refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise. As used herein, “comprises” means “includes.” Thus, “comprising A or B,” means “including A, B, or A and B,” without excluding additional elements. Dates of GenBank® Accession Nos. referred to herein are the sequences available at least as early as Dec. 29, 2021. All references, including patents and patent applications, and GenBank® Accession numbers cited herein are incorporated by reference in their entireties.
Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting.
In order to facilitate review of the various aspects of the disclosure, the following explanations of specific terms are provided:
Activator: An agent or composition that increases expression of a gene or increases activity of a gene product. In some aspects, an activator of PDE4D3 increases activity of a PDE4D3 protein (for example, increases the phosphodiesterase activity of PDE4D3). In some examples, PDE4D3 activity is increased by at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500% relative to a control, such as PDE4D3 activity prior to or in the absence of treatment with the activator. In other aspects, an activator of PDE4D3 increases transcription of a PDE4D3 gene, increases translation of a PDE4D3 mRNA, or decreases degradation of a PDE4D3 mRNA or protein, thereby increasing the level of PDE4D3 protein in the subject or target cell (such as an adipocyte). In some examples, the level of PDE4D3 protein is increased at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500% relative to a control, such as the accumulation or level of PDE4D3 protein prior to or in the absence of treatment with the activator.
Adipocyte: A type of cell that is the primary constituent of adipose tissue. These cells specialize in storing energy as fat. In addition, adipocytes are considered to be an endocrine organ that has a significant impact on the metabolism of other tissues, including regulating appetite, insulin sensitivity and immunological responses (Ali et al., Eur J Cell Biol 92 (6-7): 229-236, 2013). Adipocytes, also known as lipocytes and fat cells, are derived from mesenchymal stem cells, which differentiate to adipocytes through adipogenesis.
Administration: To provide or give a subject an agent, such as a small molecule activator of PDE4D3, or a nucleic acid molecule/vector encoding PDE4D3, by any effective route. Exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intraosseous, intramuscular, intradermal, intraperitoneal, intravenous, intrathecal, and intratumoral), sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes.
Agent: Any substance, compound or drug that is useful for achieving a particular outcome. For example, the agent can be a substance, compound, or drug capable of modulating (such as increasing) expression or activity of PDE4D3. In some aspects, the agent is a small molecule activator of PDE4D3. In other aspects, the agent is a nucleic acid molecule or vector encoding a PDE4D3 protein.
Antibody: A polypeptide ligand comprising at least one variable region that recognizes and binds (such as specifically recognizes and specifically binds) an epitope of an antigen, such as FGFR1b. Mammalian immunoglobulin molecules are composed of a heavy (H) chain and a light (L) chain, each of which has a variable region, termed the variable heavy (VH) region and the variable light (VL) region, respectively. Together, the VH region and the VL region are responsible for binding the antigen recognized by the antibody. There are five main heavy chain classes (or isotypes) of mammalian immunoglobulin, which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Antibody isotypes not found in mammals include IgX, IgY, IgW and IgNAR. IgY is the primary antibody produced by birds and reptiles, and has some functionally similar to mammalian IgG and IgE. IgW and IgNAR antibodies are produced by cartilaginous fish, while IgX antibodies are found in amphibians.
Antibody variable regions contain “framework” regions and hypervariable regions, known as “complementarity determining regions” or “CDRs.” The CDRs are primarily responsible for binding to an epitope of an antigen. The framework regions of an antibody serve to position and align the CDRs in three-dimensional space. The amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known numbering schemes, including those described by Kabat et al. (Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991; the “Kabat” numbering scheme), Chothia et al. (see Chothia and Lesk, J Mol Biol 196:901-917, 1987; Chothia et al., Nature 342:877, 1989; and Al-Lazikani et al., (JMB 273,927-948, 1997; the “Chothia” numbering scheme), and the ImMunoGene Tics (IMGT) database (see, Lefranc, Nucleic Acids Res 29:207-9, 2001; the “IMGT” numbering scheme). The Kabat and IMGT databases are maintained online.
A “single-domain antibody” refers to an antibody having a single domain (a variable domain) that is capable of specifically binding an antigen, or an epitope of an antigen, in the absence of an additional antibody domain. Single-domain antibodies include, for example, VH domain antibodies, VNAR antibodies, camelid VHH antibodies, and VL domain antibodies. VNAR antibodies are produced by cartilaginous fish, such as nurse sharks, wobbegong sharks, spiny dogfish and bamboo sharks. Camelid VHH antibodies are produced by several species including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies that are naturally devoid of light chains.
A “monoclonal antibody” is an antibody produced by a single clone of lymphocytes or by a cell into which the coding sequence of a single antibody has been transfected. Monoclonal antibodies are produced by methods known to those of skill in the art. Monoclonal antibodies include humanized monoclonal antibodies.
A “chimeric antibody” has framework residues from one species, such as human, and CDRs (which generally confer antigen binding) from another species.
A “humanized” antibody is an immunoglobulin including a human framework region and one or more CDRs from a non-human (for example a mouse, rabbit, rat, shark or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is termed a “donor,” and the human immunoglobulin providing the framework is termed an “acceptor.” In one aspect, all CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, such as about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions.
Blood glucose: Refers to the amount of glucose present in a subject's blood. A normal blood glucose level is generally considered to range from about 80 to about 130 mg/dL (if measured before a meal). If measured within two hours of a meal, blood glucose of less than about 180 mg/dL is considered normal. Higher levels of blood glucose can be indicative of diabetes or pre-diabetes. Blood glucose can be measured, for example, using a continuous glucose monitor (CGM). Blood glucose is also referred to as blood sugar.
Diabetes mellitus: A group of metabolic diseases in which a subject has high blood sugar, either because the pancreas does not produce enough insulin, or because cells do not respond to the insulin that is produced. Type 1 diabetes results from the body's failure to produce insulin. This form has also been called “insulin-dependent diabetes mellitus” (IDDM) or “juvenile diabetes”. Type 2 diabetes (T2D) results from insulin resistance, a condition in which cells fail to use insulin properly, sometimes combined with an absolute insulin deficiency. This form is also called “non-insulin-dependent diabetes mellitus” (NIDDM) or “adult-onset diabetes.” The defective responsiveness of body tissues to insulin is believed to involve the insulin receptor. Diabetes mellitus is characterized by recurrent or persistent hyperglycemia, and in some examples diagnosed by demonstrating any one of:
Effective amount or therapeutically effective amount: The amount of an agent, such as an activator of PDE4D3 or a nucleic acid molecule encoding PDE4D3, that is sufficient to prevent, treat (including prophylaxis), reduce, and/or ameliorate the symptoms and/or underlying causes of any disorder or disease. In one aspect, a “therapeutically effective amount” is sufficient to reduce or eliminate a symptom of T2D, for example by lowering blood glucose. In some examples, blood glucose is reduced by about 5%, about 10%, about 20%, about 30%, about 40% or about 50% relative to blood glucose prior to treatment.
In some examples, a “therapeutically effective amount” is the amount necessary to increase activity or expression of PDE4D3 at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500% or more compared to the activity or expression of a suitable control. In some examples, the therapeutically effective amount is the amount necessary to increase the amount of PDE4D3 protein in a cell (such as an adipocyte) by at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500% or more compared to a suitable control.
Fibroblast Growth Factor 1 (FGF1): Includes FGF1 nucleic acid molecules and proteins (e.g., OMIM 13220). FGF1 is a protein that binds to FGF receptors and is also known as the acidic FGF. FGF1 sequences are publicly available, for example from the GenBank® sequence database (e.g., Accession Nos. NP_00791 and NP_034327 provide exemplary FGF1 protein sequences, while Accession Nos. NM_000800 and NM_010197 provide exemplary FGF1 nucleic acid sequences). A skilled person can identify additional FGF1 nucleic acid and protein sequences, including FGF1 variants.
A native FGF1 sequence is one that does not include a mutation that alters the normal activity of the protein. A mature FGF1 refers to an FGF1 peptide or protein product and/or sequence following any post-translational modifications. A mutated FGF1 is a variant of FGF1 with different or altered biological activity, such as reduced mitogenicity. In one example, such a variant includes an N-terminal truncation and/or one or more additional point mutations, such as changes that decrease mitogenicity of FGF1, alter the heparin binding affinity of FGF1, and/or the thermostability of FGF1. Specific exemplary FGF1 mutant proteins are shown in
Fibroblast Growth Factor Receptor 1b (FGFR1b): A member of the FGFR1 family found on the cell surface that has tyrosine kinase activity. The FGFR1 gene can be alternatively spliced to generate distinct mRNAs, which code for two FGFR1 isoforms: FGFR1b and FGFR1c. FGFR1b sequences are publicly available, for example from GenBank® sequence database (e.g., Accession No. ACO38646.1 (SEQ ID NO: 22) provides an exemplary FGFR1b protein sequence, while Accession No. FJ809917 (SEQ ID NO: 21) provides an exemplary FGFR1b nucleic acid sequence). A skilled person can identify additional FGFR1b nucleic acid and protein sequences, including FGFR1b variants.
Gene editing: Refers to methods that enable the specific alteration (such as by deletion, insertion or substitution) of the genome of an organism. Gene editing techniques typically employ nucleases that introduce breaks into DNA strands, which allows for the removal of existing DNA and/or insertion of a desired DNA sequence (such as DNA encoding PDE4D3). Examples include, but are not limited to, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9), transcription activator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs) (see, e.g., Fridovich-Keil, “Gene Editing,” Encyclopedia Britannica, Jun. 4, 2019, available online at britannica.com/science/gene-editing; Knott and Doudna, Science 361 (6405): 866-869, 2018; U.S. Pat. No. 10,000,772; and U.S. Publication Nos. 2021/0340568, 2021/0338815, 2021/0292769, 2021/0222171). In one example, RNA (such as PDE4D3 RNA) is edited, for example using a Cas13d protein (e.g., see WO 2019/040664).
Increasing expression or activity of PDE4D3: As used herein, an agent that increases expression or activity of PDE4D3 is a compound that increases the level of PDE4D3 mRNA or PDE4D3 protein in a cell or tissue, or increases one or more activities of the PDE4D3 protein. In some examples, increasing expression of PDE4D3 includes increasing transcription of the PDE4D3 gene, increasing translation of the PDE4D3 mRNA, decreasing degradation of the PDE4D3 protein and/or increasing stability of the PDE4D3 protein, thereby increasing the level of PDE4D3 protein in a subject or a cell (such as an adipocyte) as compared to a suitable control. In other examples, increasing activity of the PDE4D3 protein includes increasing phosphodiesterase activity of PDE4D3, which results in a decrease in intracellular cAMP levels.
In some aspects, expression of PDE4D3 is increased by at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500% relative to a control. In specific non-limiting examples, a vector encoding PDE4D3 is used to express PDE4D3 in adipocytes, thereby increasing expression of PDE4D3 by at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500% relative to a control. In some aspects, activity of PDE4D3 is increased by at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 75%, at least 80%, least 90%, or at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500% relative to a control. In specific non-limiting examples, an activator of PDE4D3 (such as MR-L2) increases phosphodiesterase activity of PDE4D3 by at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500% relative to a control.
Incretins: A group of metabolic hormones released following nutrient intake. Incretins stimulate insulin secretion from pancreatic beta cells. Examples of incretin hormones include gastric inhibitory peptide (GIP; NCBI Gene ID 2695) and glucagon-like peptide-1 (GLP-1). The receptors for GIP (GIPR) and GLP-1 (GLP-1R) are expressed in adipose and other types of tissue (Capozzi et al., Endocrine Reviews 39 (5): 719-738, 2018).
Isolated: An “isolated” biological component (such as a PDE4D3 protein, or nucleic acid molecule encoding such) has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, such as other chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acid molecules and proteins which have been “isolated” thus include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acid molecules and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids. A purified or isolated cell, protein, or nucleic acid molecule can be at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure.
Linker: A bi-functional molecule that can be used to link two molecules into one contiguous molecule, for example, to link an effector molecule (such as a small molecule or drug) to a targeting molecule (such as an antibody or incretin). The terms “conjugating,” “joining,” “bonding,” or “linking” can refer to making two molecules into one contiguous molecule; for example, linking two polypeptides into one contiguous polypeptide, or covalently attaching an effector molecule or detectable marker to a polypeptide. The linkage can be either by chemical or recombinant means. “Chemical means” refers to a reaction between the antibody moiety and the effector molecule such that there is a covalent bond formed between the two molecules to form one molecule. In some aspects, the linker is an acid-sensitive linker (also known as a “pH-sensitive linker” or “acid-labile linker”). Acid-sensitive linkers include a chemical bond, such as an imine, hydrazone, oxime, amide, acetal, or orthoester bond, that is cleaved under acidic conditions (see, e.g., Zhuo et al., Molecules 25:5649, 2020). In particular examples, the acid-sensitive linker is a carbonate, hydrazone or silyl ether, such as:
Mammal: This term includes both human and non-human mammals. Similarly, the term “subject” includes both human and veterinary subjects (such as cats, dogs, cows, and pigs) and rodents (such as mice and rats). In some examples, the mammal has or is at risk of developing type 2 diabetes.
MR-L2: A small molecule activator of PDE4 long isoform variants, such as PDE4D3. The chemical name of MR-L2 is 2-(3-(4-chloro-3-fluorophenyl)-5-ethyl-1H-1,2,4-triazol-1-yl)-N-(3,5-dichlorobenzyl) acetamide. The chemical structure of MR-L2 is shown below (see Omar et al., Proc Natl Acad Sci USA 116 (27): 13320-13329, 2019):
Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence (such as a PDE4D3 coding sequence). Generally, operably linked DNA sequences are contiguous and, where necessary, join two protein coding regions, in the same reading frame.
Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers of use are conventional. Remington: The Science and Practice of Pharmacy, 22nd ed., London, UK: Pharmaceutical Press, 2013), describes compositions and formulations suitable for pharmaceutical delivery of the agents described herein. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
Phosphodiesterase 4D (PDE4D): One of four mammalian counterparts to the fruit fly ‘dunce’ gene. PDE4D proteins have 3′,5′-cyclic-AMP phosphodiesterase activity and degrade cAMP, which acts as a signal transduction molecule in multiple cell types. The PDE4D gene uses different promoters to generate multiple alternatively spliced transcript variants that encode functional proteins (e.g., PDE4D1, PDE4D2, PDE4D3, PDE4D4, PDE4D5, PDE4D6, PDE4D7, PDE4D8 and PDE4D9; see NCBI Gene ID 5144). Nucleic acid and protein sequences for multiple PDE4D isoforms are publicly available, such as under NCBI Gene ID 5144. Exemplary PDE4D3 isoform sequences include GenBank Accession Nos. NM_006203.5 (nucleotide; SEQ ID NO: 1) and NP_006194.2 (protein; SEQ ID NO: 2). In some examples, a PDE4D3 nucleic acid sequence has at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1, and encodes a protein having PDE4D3 biological activity. In some examples, a PDE4D3 protein has at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2, and has PDE4D3 biological activity. In some example, such a PDE4D3 does not include a substitution at position 44, such as an S44A substitution.
Preventing, treating or ameliorating a disease: “Preventing” a disease refers to inhibiting the full development of a disease. “Treating” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition (such as T2D) after it has begun to develop (such as lowering blood glucose). “Ameliorating” refers to the reduction in the number or severity of signs or symptoms of a disease.
Promoter: An array of nucleic acid control sequences which direct transcription of a nucleic acid. A promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription. In some aspects, the promoter is an adipose/adipocyte-specific promoter, such as the adiponectin promoter/enhancer (see, e.g., Wang et al., Endocrinology 151 (6): 2933-2939, 2010). In specific examples, the promoter is the adiponectin promoter/enhancer of SEQ ID NO: 27.
Sequence identity: The similarity between amino acid or nucleotide sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences. Homologs or variants of a polypeptide will possess a relatively high degree of sequence identity when aligned using standard methods.
Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research 16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994, presents a detailed consideration of sequence alignment methods and homology calculations.
The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. A description of how to determine sequence identity using this program is available on the NCBI website on the internet.
Subject: Any mammal, such as humans, non-human primates, pigs, sheep, cows, dogs, cats, rodents and the like which is to be the recipient of a particular treatment, such as treatment with an agent that increases expression or activity of PDE4D3, as described herein. In some examples, the subject is a human, for example a human subject with or at risk of T2D.
Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replicate in the host cell, such as an origin of replication. A vector may also include one or more PDE4D3 coding sequences, and/or selectable marker genes (such as antibiotic resistance or an enzyme) and other genetic elements. A vector can transduce, transform, or infect a cell, thereby causing the cell to express nucleic acids and/or proteins other than those native to the cell. A vector optionally includes materials to aid in achieving entry of the nucleic acid into the cell, such as a viral particle, liposome, protein coating, or the like. In some aspects, the vector is an adeno-associated virus (AAV) vector, an adenovirus (Ad) vector, or a lentivirus vector. In some aspects, the vector is a plasmid.
Adipose tissue holds 80-85% of the body's energy reserves, hence the decision to store or release is central to physiological homeostasis. However, while adipose lipolysis is triggered by multiple factors, insulin is the only known anti-lipolytic regulator. Disclosed herein is a signaling cascade induced by FGF1 that functions as an alternate lipolytic suppressive pathway and establishes FGF1 as a regulator of fatty acid homeostasis. FGF1 is an essential mediator of adipose remodeling (Jonker et al., 2012) and rapidly normalizes blood glucose levels in diabetic mouse models in an adipose FGFR1-dependent manner (Suh et al., 2014). The present disclosure demonstrates that FGF1-FGFR1 signaling reduces blood glucose levels by suppressing adipose lipolysis and thereby, decreases HGP through the allosteric regulation of pyruvate carboxylase. Within the adipocyte, FGF1 engages an alternate PI3K/PDE4D circuit to inhibit the cAMP-PKA axis. The resultant reductions in cAMP decrease PKA-mediated phosphorylation of HSL and its subsequent translocation to the lipid droplet. The PDE4D dependency of the anti-lipolytic and anti-diabetic actions of FGF1 suggest a parallel pathway to the established insulin-PDE3B axis (
Mutation of S85 (QRRES motif, SEQ ID NO: 36), which enhances in vitro PDE4D3 activity (Sette and Conti, 1996), had a minor effect on suppression of lipolysis when PDE4D3-S85A was overexpressed in adipocytes. In contrast, mutation of S44 (FRRHS motif; SEQ ID NO: 37) abrogated the suppression of lipolysis by PDE4D3 despite overexpression, indicating a novel regulatory role for this phosphorylation site. In support of this, over-expression of WT and S44A PDE4D3 resulted in similar PDE activities in adipocytes. The N-terminus of PDE4D3 and its PKA phosphorylation site are implicated in the interaction of PDE4D3 with the muscle-specific A-kinase anchoring protein (mAKAP) (Carlisle Michel et al., 2004; Dodge et al., 2001). Hence, this site may be pivotal in compartmentalization of PKA-PDE4D3 complexes. In addition, binding of phosphatidic acid to the N-terminal sequence activates PDE4 (Grange et al., 2000).
While the present disclosure describes the action of FGF1 in adipose tissue, central administration of a single FGF1 dose can also restore glucose homeostasis in diabetic models, however the kinetics are slow (weeks). The improvement in blood glucose levels in the Zucker T2D diabetic rat model was attributed to preservation of β cell function and increased hepatic glucose uptake, whereas a suppression of the hypothalamic-pituitary-adrenal (HPA) axis resulting in lower lipolysis and hepatic glucose output was described in T1D rats (Perry et al., 2015b; Scarlett et al., 2019; Scarlett et al., 2016). While the rapid glucose lowering seen with peripheral FGF1 delivery is consistent with lipolytic regulation, the delayed effects of central FGF1 delivery suggest a distinct mechanism.
The findings disclosed herein that implicate adipose PDE4D in the beneficial actions of FGF1 appear at odds with the anti-diabetic effects of systemic PDE4 inhibition (Möllmann et al., 2017; Vollert et al., 2012). Increased GLP1 secretion, higher serum insulin levels, and increased muscle mitochondrial function are associated with chronic PDE4 inhibition indicating adipose tissue independent effects. (Ong et al., 2009; Park et al., 2012; Vollert et al., 2012). In addition, contributions of the associated weight loss and potential anti-inflammatory effects of PDE4B inhibition to improvements in glucose tolerance and fasting glucose levels are not known (Jin and Conti, 2002; Komatsu et al., 2013; Möllmann et al., 2017; Zhang et al., 2009). In contrast to the pleiotropic effects of systemic inhibition, the present study indicates that PDE4D activity in adipocytes is necessary for FGF1 to suppress lipolysis and lower blood glucose levels (
PDE3B is important for controlling cAMP levels and lipolysis in adipocytes, however PDE4 accounts for approximately half of the total PDE activity in white adipocytes (Young et al., 2006). Moreover, maximal adipose lipolysis requires both PDE3 and PDE4 inhibitors, supporting an underappreciated role of PDE4 in the regulation of lipolysis (DiPilato et al., 2015b; Kraynik et al., 2013; Snyder et al., 2005). In addition, the finding that FGF1 enhances PDE4D3 phosphorylation at S44 correlates with the post-prandial increases seen at this site in both chow and HFD mice (
The anti-lipolytic effect of FGF1 in the fed state contrasts to FGF21-induced lipolysis in the fasted state. Accordingly, these findings implicate an unexpected FGF1: FGF21 molecular balance regulating the storage and release of fat in the fed and fasted state, respectively. This physiologic paradigm describes a mechanism that not only manages glucose homeostasis in health, but via FGF1 injection, can be used to quickly rebalance glucose levels in insulin resistant type 2 diabetes. Thus, in addition to a new signaling cascade that suppresses lipolysis, these findings reveal the therapeutic potential of the FGF1-PDE4D axis in diabetes.
Described herein is the finding that fibroblast growth factor 1 (FGF1) suppresses adipose lipolysis and the anti-lipolytic activities of FGF1 are required for acute glucose lowering. Also described is the finding that these FGF1 activities are mediated by the activation of phosphodiesterase 4D isoform 3 (PDE4D3) in adipose tissue. In view of these findings, the present disclosure provides methods of lowering blood glucose and/or treating T2D in a subject by increasing expression or activity of PDE4D3. In some examples, blood glucose is lowered by at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, or at least 75%, for example relative to an amount prior to treatment or an amount without use of the disclosed methods. In some examples, phosphodiesterase 4D isoform 3 (PDE4D3) activity and/or expression is increased using the disclosed methods by at least 10%, at least 20%, at least 25%, at least 50%, at least 75%, at least 90%, at least 100%, at least 200%, or at least 500%, for example relative to an amount prior to treatment with the agent that increases expression or activity of PDE4D3 or an amount without use of the agent that increases expression or activity of PDE4D3.
Provided herein is a method of reducing blood glucose in a subject. In some aspects, the method includes administering to the subject a therapeutically effective amount of an agent that increases expression or activity of PDE4D3 in adipocytes of the subject. In some examples, the subject has been diagnosed with T2D, or is at high risk of developing T2D.
Also provided herein is a method of treating T2D in a subject. In some aspects, the method includes administering to the subject a therapeutically effective amount of an agent that increases expression or activity of PDE4D3 in adipocytes of the subject.
Also provided are agents that can be used in such methods, such as a nucleic acid molecule encoding a PDE4D3 protein operably linked to an adipocyte-specific promoter, and vectors (such as plasmids or viral vectors) that include such. In one example the agent is a small molecule activator of PDE4D3 conjugated to an antibody that specifically binds fibroblast growth factor receptor 1b (FGFR1b), such as an antibody disclosed in US 2012/0121609. In one example the agent is a small molecule activator of PDE4D3 conjugated to an incretin, such as GIP or GLP-1. Compositions that include such agents are also provided, for example which may further include a pharmaceutically acceptable carrier such as water, saline, or a buffer.
In some aspects, the agent that increases expression or activity of PDE4D3 includes a nucleic acid molecule (e.g., DNA, cDNA, or RNA) encoding a PDE4D3 protein. In some examples, the nucleic acid molecule encoding the PDE4D3 protein is operably linked to an adipocyte-specific promoter and/or enhancer, such as the adiponectin promoter and/or enhancer. In particular non-limiting examples, the adiponectin promoter/enhancer includes SEQ ID NO: 27. In some examples, the nucleic acid molecule further includes a target sequence for miR-122a, such as SEQ ID NO: 28, to restrict expression to adipose tissue.
In some aspects, the nucleic acid molecule encoding the PDE4D3 protein includes a vector. In some examples, the vector is a viral vector, such as but not limited to, an adenovirus vector, an adeno-associated virus (AAV) vector, or a lentivirus vector. In other examples, the vector is a plasmid vector.
In other aspects, the nucleic acid molecule encoding the PDE4D3 protein is introduced into adipocytes using a gene editing method. Gene editing methods are known and include, for example, CRISPR-Cas9, Cas13d, TALENs, or ZFNs. Gene editing techniques are known, such as those described in U.S. Pat. No. 10,000,772; and U.S. Publication Nos. 2021/0340568, 2021/0338815, 2021/0292769, 2021/0222171.
In some aspects of the disclosed methods, the PDE4D3 protein includes the amino acid sequence set forth as SEQ ID NO: 1, or a variant thereof having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, or 100% identity to SEQ ID NO: 1. In some examples, such a protein includes a native Ser at position 44. In some examples, such a protein does not include a S44A substitution.
In some aspects, the agent that increases expression or activity of PDE4D is a small molecule activator of PDE4D3. In some examples, the small molecule activator of PDE4D3 is an N-substituted-2-(3-aryl-1H-1,2,4-triazol-1-yl) acetamide. In specific non-limiting examples, the small molecule activator of PDE4D3 is 2-(3-(4-chloro-3-fluorophenyl)-5-ethyl-1H-1,2,4-triazol-1-yl)-N-(3,5-dichlorobenzyl) acetamide (also known as MR-L2).
In some examples, the small molecule activator of PDE4D3 is conjugated to an antibody that specifically binds fibroblast growth factor receptor 1b (FGFR1b), such as a polyclonal FGFR1b antibody, a monoclonal FGFR1b antibody, or fragment of an FGFR1b antibody (such as an antibody provided in US 2012/0121609). In other examples, the small molecule activator of PDE4D3 is conjugated to an incretin, such as but not limited to, gastric inhibitory peptide (GIP) or glucagon-like peptide-1 (GLP-1), such as a peptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, or 100% identity to SEQ ID NO: 24 or 26. In particular examples, the small molecule activator of PDE4D3 is conjugated to the antibody or the incretin via an acid-sensitive linker.
The agent that increases expression or activity of PDE4D3, or a pharmaceutical composition thereof, can be administered to a subject, such as a mammalian subject (e.g., a human subject), by any means, including orally, intravenously, intramuscularly, intraperitoneally, intranasally, intradermally, intrathecally, subcutaneously, via inhalation or via suppository. In one non-limiting example, the composition is administered via injection. In some examples, site-specific administration of the composition can be used, for example by administering the agent to adipose tissue (for example by using a pump, or by implantation of a slow release form at the adipose depot). The particular mode of administration and the dosage regimen will be selected by the attending clinician, taking into account the particulars of the case (e.g., the subject, the disease, the disease state involved, the particular treatment, and whether the treatment is prophylactic). Treatment can involve daily or multi-daily or less than daily (such as weekly, every other week, monthly, every 7 days, every 10 days, every 14 days, every 21 days, every 30 days, every 40 days, every 60 days, etc.) doses of the agent or pharmaceutical composition over a period of a few days, few weeks, to months, or even years. For example, a therapeutically effective amount of the agent can be administered in a single dose, once daily, twice daily, three times daily, four times daily, six times daily, weekly, every other week, every three weeks, every month, every other month, or in several doses, for example daily, or during a course of treatment. In a particular non-limiting example, treatment involves once daily dose, twice daily dose, once weekly dose, every other week dose, or monthly dose.
When a viral vector is utilized for administration of a nucleic acid encoding a PDE4D3 protein (such as the PDE4D3 protein of SEQ ID NO: 2, or a protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2), the recipient can receive a dose in the range of from about 105 to about 1010 plaque forming units/mg, although a lower or higher dose can be administered. Examples of methods for administering the composition to a subject (such as a mammalian subject) include, but are not limited to, exposure of cells to the recombinant virus ex vivo, or injection of the composition into the affected tissue or intravenous, subcutaneous, intradermal, or intramuscular administration of the virus. Alternatively, the viral vector may be administered locally by direct injection into adipose tissue in a pharmaceutically acceptable carrier. Generally, the quantity of viral vector carrying the PDE4D3 nucleic acid sequence to be administered (such as SEQ ID NO: 1, or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1), is based on the titer of virus particles. An exemplary range to be administered is 105 to 1010 virus particles.
In some aspects, the methods further include administering to the subject a therapeutically effective amount of a mature FGF1 protein or a modified mature FGF1 protein. In some examples, the modified mature FGF1 protein has reduced mitogenicity and/or increased stability compared to native FGF1 protein, and/or the modified mature FGF1 protein selectively binds FGFR1b. Exemplary FGF1 proteins (including mutant FGF1 proteins) are described in, for example, WO 2015/061331, WO 2015/061351, WO 2015/061361, WO 2011/130729, WO 2016/172153, WO 2016/172156, WO 2016/172290, WO 2018/026713, WO 2018/112200 and US 2021/0032303. In specific examples, the mutant FGF1 protein is one shown in
In some aspects, the method further includes administering a therapeutically effective amount of an additional therapeutic agent, such as an agent useful in the treatment of diabetes. Anti-diabetic agents are generally categorized into six classes: biguanides (e.g., metformin); thiazolidinediones (including rosiglitazone (Avandia®), pioglitazone (Actos®), rivoglitazone, and troglitazone); sulfonylureas; inhibitors of carbohydrate absorption; fatty acid oxidase inhibitors and anti-lipolytic drugs; and weight-loss agents. Any of these agents can be used in the methods disclosed herein. In some examples, the additional therapeutic agent is insulin, an alpha-glucosidase inhibitor, amylin agonist, dipeptidyl-peptidase 4 (DPP-4) inhibitor, meglitinide, sulfonylurea, or a peroxisome proliferator-activated receptor (PPAR)-gamma agonist. In particular examples, the PPAR-gamma agonist is a thiazolidinedione (such as pioglitazone, rosiglitazone, rivoglitazone, or troglitazone), aleglitazar, farglitazar, muraglitazar, or tesaglitazar.
In some examples, an agent that increases expression or activity of PDE4D3 is administered in combination with effective doses of one or more FGF1 proteins (such as mutant FGF1 proteins, e.g., a mutant shown in
In some aspects, the subject is a mammal. In some examples, the subject is a human, dog or cat, such as a human, dog or cat with elevated blood glucose. In particular non-limiting examples, the subject is a human who has or is at risk of T2D.
In some aspects, the method further includes selecting a subject with elevated blood glucose, selecting a subject who is at risk of T2D, selecting a subject who has pre-diabetes, or selecting a subject who has T2D.
In some examples, a subject with diabetes may be clinically diagnosed by a fasting plasma glucose (FPG) concentration of greater than or equal to 7.0 millimole per liter (mmol/L) (126 milligram per deciliter (mg/dL)), or a plasma glucose concentration of greater than or equal to 11.1 mmol/L (200 mg/dL) at about two hours after an oral glucose tolerance test (OGTT) with a 75 gram (g) load, or in a patient with classic symptoms of hyperglycemia or hyperglycemic crisis, a random plasma glucose concentration of greater than or equal to 11.1 mmol/L (200 mg/dL), or HbA1c levels of greater than or equal to 6.5%. In other examples, a subject with pre-diabetes may be diagnosed by impaired glucose tolerance (IGT). An OGTT two-hour plasma glucose of greater than or equal to 140 mg/dL and less than 200 mg/dL (7.8-11.0 mM), or a fasting plasma glucose (FPG) concentration of greater than or equal to 100 mg/dL and less than 125 mg/dl (5.6-6.9 mmol/L), or HbA1c levels of greater than or equal to 5.7% and less than 6.4% (5.7-6.4%) is considered to be IGT, and indicates that a subject has pre-diabetes. Additional information can be found in Standards of Medical Care in Diabetes—2010 (American Diabetes Association, Diabetes Care 33: S11-61, 2010).
In some examples, treating T2D includes one or more of increasing glucose tolerance (such as an increase of at least 5%, at least 10%, at least 20%, or at least 50%, for example relative to no administration of the agent), decreasing insulin resistance (for example, decreasing plasma glucose levels, decreasing plasma insulin levels, or a combination thereof, such as decreases of at least 5%, at least 10%, at least 20%, or at least 50%, for example relative to no administration of the agent), decreasing serum triglycerides (such as a decrease of at least 10%, at least 20%, or at least 50%, for example relative to no administration of the agent), decreasing free fatty acid levels (such as a decrease of at least 5%, at least 10%, at least 20%, or at least 50%, for example relative to no administration of the agent), and decreasing HbA1c levels in the subject (such as a decrease of at least 0.5%, at least 1%, at least 1.5%, at least 2%, or at least 5% for example relative to no administration of the agent). In some aspects, the disclosed methods include measuring glucose tolerance, insulin resistance, plasma glucose levels, plasma insulin levels, serum triglycerides, free fatty acids, and/or HbA1c levels in a subject.
In some aspects of the methods disclosed herein, a nucleic acid molecule encoding a PDE4D3 protein is administered to a subject. Based on the genetic code, nucleic acid sequences coding for PDE4D3 protein can be routinely generated. In some examples, such a sequence is optimized for expression in particular host cells, such as mammalian host cells (e.g., human cells). Thus, in some aspects, the agents disclosed herein include a nucleic acid molecule encoding a PDE4D3 protein.
In some aspects, the amino acid sequence of the PDE4D3 protein is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 2. A nucleic acid sequence that codes for a PDE4D3 protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2 can readily be produced using the PDE4D3 amino acid sequence provided herein (or publicly available PDE4D3 protein sequences), and the genetic code. In addition, a variety of clones containing functionally equivalent nucleic acids, such as nucleic acids which differ in sequence but which encode the same PDE4D3 protein sequence, can be generated. In some examples, the nucleic acid molecule includes SEQ ID NO: 1, or a variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1.
Nucleic acid molecules include DNA, cDNA, and RNA sequences which encode a PDE4D3 protein. Silent mutations in the coding sequence result from the degeneracy (i.e., redundancy) of the genetic code, whereby more than one codon can encode the same amino acid residue. Thus, for example, leucine can be encoded by CTT, CTC, CTA, CTG, TTA, or TTG; serine can be encoded by TCT, TCC, TCA, TCG, AGT, or AGC; asparagine can be encoded by AAT or AAC; aspartic acid can be encoded by GAT or GAC; cysteine can be encoded by TGT or TGC; alanine can be encoded by GCT, GCC, GCA, or GCG; glutamine can be encoded by CAA or CAG; tyrosine can be encoded by TAT or TAC; and isoleucine can be encoded by ATT, ATC, or ATA. Tables showing the standard genetic code can be found in various sources (see, for example, Stryer, 1988, Biochemistry, 3rd Edition, W.H. 5 Freeman and Co., NY).
Codon preferences and codon usage tables for a particular species can be used to engineer isolated nucleic acid molecules encoding a PDE4D3 protein (such as one encoding a protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2) that take advantage of the codon usage preferences of that particular species. For example, the proteins disclosed herein can be designed to have codons that are preferentially used by a particular organism of interest, such as humans or other mammals.
In some examples, the nucleic acid encodes a PDE4D3 protein having a native Ser at position 44. In some examples, such a PDE4D3 protein does not include a S44A substitution.
A nucleic acid encoding the desired protein can be cloned or amplified by in vitro methods, such as the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self-sustained sequence replication system (3SR) and the Qβ replicase amplification system (QB). A variety of cloning and in vitro amplification methodologies can be used. In addition, nucleic acids encoding sequences encoding a desired protein can be prepared by cloning techniques. Examples of appropriate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through cloning are found in Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring, Harbor, N.Y., 1989, and Ausubel et al., (1987) in “Current Protocols in Molecular Biology,” John Wiley and Sons, New York, N.Y.
Nucleic acid sequences encoding a PDE4D3 protein can be prepared by any suitable method including, for example, cloning of appropriate sequences or by direct chemical synthesis by methods such as the phosphotriester method of Narang et al., Meth. Enzymol. 68:90-99, 1979; the phosphodiester method of Brown et al., Meth. Enzymol. 68:109-151, 1979; the diethylphosphoramidite method of Beaucage et al., Tetra. Lett. 22:1859-1862, 1981; the solid phase phosphoramidite triester method described by Beaucage & Caruthers, Tetra. Letts. 22 (20): 1859-1862, 1981, for example, using an automated synthesizer as described in, for example, Needham-VanDevanter et al., Nucl. Acids Res. 12:6159-6168, 1984; and, the solid support method of U.S. Pat. No. 4,458,066. Chemical synthesis produces a single stranded oligonucleotide. This can be converted into double stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template. One of skill would recognize that while chemical synthesis of DNA is generally limited to sequences of about 100 bases, longer sequences may be obtained by the ligation of shorter sequences.
In some aspects herein, the PDE4D3 nucleic acid coding sequence (such as a sequence encoding SEQ ID NO: 2, or encoding a protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2, which in some examples does not have an S44A substitution) can be inserted into an expression vector including, but not limited to a plasmid, virus or other vehicle that can be manipulated to allow insertion or incorporation of sequences and can be expressed in, for example, a mammalian (such as human) host.
Nucleic acid sequences encoding a PDE4D3 protein (such as a nucleic acid encoding SEQ ID NO: 2, or encoding a protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2) can be operatively linked to expression control sequences. An expression control sequence operatively linked to a desired protein coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences. The expression control sequences include, but are not limited to appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a PDE4D3 protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. In some examples, the nucleic acid sequence is operably linked to a promoter and/or enhancer that allows for specific expression in adipocytes and/or adipose tissue. In specific examples, the adipocyte-specific promoter/enhancer is an adiponectin promoter/enhancer. In other specific examples, the nucleic acid molecule includes (or further includes) a target sequence for an adipose-specific microRNA, such as miR-122a. In specific instances, the miR-122a target sequences includes or consists of SEQ ID NO: 28.
In some aspects, the nucleic acid molecule is a viral vector that encodes a PDE4D3 protein (such as SEQ ID NO: 2, or a protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2). Exemplary viral vectors include adenovirus, adeno-associated virus (AAV), polyoma, SV40, vaccinia virus, herpes viruses (including HSV and EBV), Sindbis viruses, alphaviruses and retroviruses of avian, murine, and human origin (e.g., lentivirus vectors). Other suitable vectors include orthopox vectors, avipox vectors, fowlpox vectors, capripox vectors, suipox vectors, and poliovirus vectors. In particular examples, the viral vector is an adenovirus vector, an AAV vector or a lentivirus vector.
Viral vectors that encode a PDE4D3 protein (such as SEQ ID NO: 2, or encode a protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2) can include at least one expression control element operationally linked to the nucleic acid sequence encoding the protein. The expression control elements are inserted in the vector to control and regulate the expression of the nucleic acid sequence. Examples of expression control elements of use in these vectors includes, but is not limited to, lac system, operator and promoter regions of phage lambda, yeast promoters and promoters derived from polyoma, adenovirus, retrovirus or SV40. In some examples, the vector includes a promoter and/or enhancer that allows for specific expression in adipocytes and/or adipose tissue. In specific examples, the adipocyte-specific promoter/enhancer is an adiponectin promoter/enhancer. In some examples, the vector includes or further includes a target sequence for miR-122a, such as SEQ ID NO: 28.
Additional operational elements for a viral vector include, but are not limited to, leader sequence, termination codons, polyadenylation signals and any other sequences necessary for the appropriate transcription and subsequent translation of the nucleic acid sequence encoding the protein in the host. The expression vector can contain additional elements necessary for the transfer and subsequent replication of the expression vector containing the nucleic acid sequence in a host system. Examples of such elements include, but are not limited to, origins of replication and selectable markers. It will further be understood by one skilled in the art that such vectors are easily constructed using conventional methods (Ausubel et al., (1987) in “Current Protocols in Molecular Biology,” John Wiley and Sons, New York, N.Y.) and are commercially available.
In some aspects of the disclosed methods, the agent that increases expression or activity of PDE4D3 is a small molecule activator of PDE4D3. In some examples, the small molecule activator of PDE4D3 is an N-substituted-2-(3-aryl-1H-1,2,4-triazol-1-yl) acetamide. In particular non-limiting examples, the N-substituted-2-(3-aryl-1H-1,2,4-triazol-1-yl) acetamide is 2-(3-(4-chloro-3-fluorophenyl)-5-ethyl-1H-1,2,4-triazol-1-yl)-N-(3,5-dichlorobenzyl) acetamide (MR-L2). As described in Omar et al. (Proc Natl Acad Sci USA 116 (27): 13320-13329, 2019), MR-L2 is an allosteric activator of PDE4 long form cyclic AMP phosphodiesterases (such as PDE4D3).
In some aspects, the small molecule activator of PDE4D3 (such as MR-L2) is conjugated to a molecule that directs delivery of the small molecule to adipose tissue/adipocytes. Thus, in some aspects, the agents disclosed herein include a small molecule activator of PDE4D3, for example conjugated to a molecule that directs delivery of the small molecule to adipose tissue/adipocytes. In some examples, the small molecule is conjugated to an antibody or fragment thereof (such as an Fab fragment), such as a monoclonal antibody, that specifically binds a protein expressed on adipocytes, such as fibroblast growth factor receptor 1b (FGFR1b). Antibodies that specifically bind FGFR1b are known (see, e.g., U.S. 2012/0121609) and can be generated and/or identified using standard methods, such as by phage display or immunization of mice or rabbits.
In other examples, the small molecule activator of PDE4D3 is conjugated to an incretin, such as GIP or GLP-1. The receptors for GIP (GIPR) and GLP-1 (GLP-1R) are expressed in adipose tissue (Capozzi et al., Endocrine Reviews 39 (5): 719-738, 2018). Thus, conjugation of the small molecule activator to GIP or GLP-1, targets the molecule to adipose tissue.
In some examples, the small molecule activator (such as MR-L2) is conjugated to a monoclonal antibody (such as an FGFR1b-specific antibody) or an incretin (such as GIP or GLP-1) via an acid-sensitive linker. Upon delivery of the conjugated molecules to adipocytes, the conjugates are internalized and the small molecular activator is cleaved from the antibody or incretin within endosomes, which have a pH range of about 6.5 (early endosomes) to about 4.5 (lysosomes). Acid-sensitive linkers are well-known (see, e.g., Zhuo et al., Molecules 25:5649, 202); appropriate acid-sensitive linkers can be selected by a skilled person. In some examples, the acid-sensitive linker is a carbonate, hydrazone or silyl ether linker.
In the context of the disclosed methods, an agent that increases expression or activity of PDE4D3 (such as a nucleic acid molecule/vector encoding PDE4D3, or a small molecule activator of PDE4D3) can be administered to the subject as part of a pharmaceutical composition. Such pharmaceutical compositions can be formulated with an appropriate pharmaceutically acceptable carrier, depending upon the particular mode of administration chosen. Thus, in some aspects, the agents disclosed herein are part of a pharmaceutical composition.
In some aspects, the pharmaceutical composition consists essentially of a nucleic acid (or vector) encoding a PDE4D3 protein and a pharmaceutically acceptable carrier. In other aspects, the pharmaceutical composition consists essentially of a small molecule activator of PDE4D3 (such as MR-L2) and a pharmaceutically acceptable carrier. In these aspects, additional therapeutically effective agents are not included in the compositions.
In other aspects of the disclosed methods, the pharmaceutical composition includes one or more additional therapeutic agents, such as agents for the treatment of T2D. Thus, the pharmaceutical compositions can include a therapeutically effective amount of another agent. Examples of such agents include, without limitation, antidiabetic agents for example, insulin, metformin, sulphonylureas (e.g., glibenclamide, tolbutamide, glimepiride), nateglinide, repaglinide, thiazolidinediones (e.g., rosiglitazone, pioglitazone), peroxisome proliferator-activated receptor (PPAR)-γ agonists (such as C1262570, aleglitazar, farglitazar, muraglitazar, tesaglitazar, and TZD) and PPAR-γ antagonists, PPAR-gamma/alpha modulators (such as KRP 297), alpha-glucosidase inhibitors (e.g., acarbose, voglibose), dipeptidyl peptidase (DPP)-IV inhibitors (such as LAF237, MK-431), alpha2-antagonists, agents for lowering blood sugar, cholesterol-absorption inhibitors, 3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA) reductase inhibitors (such as a statin), insulin and insulin analogues, GLP-1 and GLP-1 analogues (e.g. exendin-4) or amylin. In some examples, the pharmaceutical composition includes, or further includes, a FGF1 protein, or a mutant FGF1 protein, such as one or more FGF1 proteins shown in
The pharmaceutically acceptable carriers and excipients useful in this disclosure are conventional. See, e.g., Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21st Edition (2005). For instance, parenteral formulations usually include injectable fluids that are pharmaceutically and physiologically acceptable fluid vehicles such as water, physiological saline, other balanced salt solutions, aqueous dextrose, glycerol or the like. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, pH buffering agents, or the like, for example sodium acetate or sorbitan monolaurate. Excipients that can be included are, for instance, other proteins, such as human serum albumin or plasma preparations.
In some aspects, an agent that increases expression or activity of PDE4D3 is included in a controlled release formulation, for example, a microencapsulated formulation. Various types of biodegradable and biocompatible polymers can be used, and methods of encapsulating a variety of synthetic compounds, proteins and nucleic acids, have been described (see, for example, U.S. Patent Publication Nos. 2007/0148074; 2007/0092575; and 2006/0246139; U.S. Pat. Nos. 4,522,811; 5,753,234; and 7,081,489; PCT Publication No. WO/2006/052285; Benita, Microencapsulation: Methods and Industrial Applications, 2nd ed., CRC Press, 2006).
In other aspects, an agent that increases expression or activity of PDE4D3 is included in a nanodispersion system. Exemplary nanodispersion systems and methods for producing such nanodispersions are provided in e.g., U.S. Pat. No. 6,780,324; U.S. Pat. Publication No. 2009/0175953. For example, a nanodispersion system includes a biologically active agent and a dispersing agent (such as a polymer, copolymer, or low molecular weight surfactant). Exemplary polymers or copolymers include polyvinylpyrrolidone (PVP), poly(
With regard to the administration of nucleic acids, one approach to administration of nucleic acids is direct treatment with plasmid DNA, such as with a mammalian expression plasmid. As described above, the nucleotide sequence encoding a PDE4D3 protein (such as encoding SEQ ID NO: 2, or encoding a protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2), can be placed under the control of a promoter, such as an adipocyte-specific promoter, to increase expression of the protein in adipose tissue.
Many types of release delivery systems can be used. Examples include polymer based systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Pat. No. 5,075,109. Delivery systems also include non-polymer systems, such as lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides; hydrogel release systems; silastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. Specific examples include, but are not limited to: (a) erosional systems in which an agent that increases expression or activity of PDE4D3, such as a PDE4D3 protein (such as SEQ ID NO: 2, or a protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2), a polynucleotide encoding such a protein, or a small molecule activator (such as MR-L2) is contained in a form within a matrix such as those described in U.S. Pat. Nos. 4,452,775; 4,667,014; 4,748,034; 5,239,660; and 6,218,371 and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Pat. Nos. 3,832,253 and 3,854,480. In addition, pump-based hardware delivery systems can be used, some of which are adapted for implantation.
Use of a long-term sustained release implant can be suitable for treatment of chronic conditions, such as diabetes. Long-term release, as used herein, means that the implant is constructed and arranged to deliver therapeutic levels of the active ingredient for at least 30 days, such as 60 days. Long-term sustained release implants include the release systems described above. These systems have been described for use with nucleic acids (see U.S. Pat. No. 6,218,371).
The dosage form of the pharmaceutical composition can be determined by the mode of administration chosen. For instance, in addition to injectable fluids, topical, inhalation, oral, and suppository formulations can be employed. Topical preparations can include eye drops, ointments, sprays, patches, and the like. Inhalation preparations can be liquid (e.g., solutions or suspensions) and include mists, sprays and the like. Oral formulations can be liquid (e.g., syrups, solutions or suspensions), or solid (e.g., powders, pills, tablets, or capsules). Suppository preparations can also be solid, gel, or in a suspension form. For solid compositions, conventional non-toxic solid carriers can include pharmaceutical grades of mannitol, lactose, cellulose, starch, or magnesium stearate.
The following examples are provided to illustrate certain particular features and/or aspects. These examples should not be construed to limit the disclosure to the particular features or aspects described.
This example describes the materials and methods used for the studies described in Examples 2-6.
Mice were kept in 12 h light/dark cycle in a temperature-controlled environment. Mice had free access to food and water unless noted otherwise. C57/B6 mice background was used unless noted otherwise. C57BL/6J and ob/ob (000632-B6. Cg-Lepob/J) male mice were obtained from Jackson Laboratory. PDE4D knock-out mice (034588-UCD) were obtained from MMRRC. For diet-induced obesity (DIO) studies, mice were fed a high fat diet (HFD) (60% fat, F3282; Bio-Serv) for minimum of 12 weeks to induce insulin resistance when diet-induced obese model is used. Insulin levels and blood glucose levels were monitored to confirm insulin resistance. To generate adipose-specific FGFR1 deletion, adiponectin-cre mice (B6; FVB-Tg (Adipoq-cre)1Evdr/J; Stock: 010803) were crossed to Fgfr1 flox/flox (B6.129S4-Fgfr1tm5.1Sor/J; Stock: 00767) mice. For AAV mediated expression of target proteins in adipose tissue, 5×10E+11 genomic copies of AAV were injected via tail vein or retro-orbital route.
All cells were grown at 37° C. in a 5% CO2 humid atmosphere in DMEM, 10% FBS (GemCell, 100-500), 1× Antibiotic-Antimycotic (Gibco, 15240096) unless indicated otherwise. 3T3-L1 cells (ATCC, CL-173), mouse SVF-derived cells and human pre-adipocytes (Promocell, C-12735) were differentiated and treated as described below.
Recombinant FGF1 (rFGF1) was dissolved in phosphate-buffered saline (PBS) at a concentration of 0.2 mg/ml and was injected subcutaneously at a 0.5 mg kg−1 dose. Blood glucose was monitored at indicated time points after injection. For initial measurements (2-4 h) food was removed after injection to exclude any indirect effects stemming from anorexigenic effects of FGF1. PDE4 inhibitor roflumilast stock in DMSO was diluted at 1 mg/ml in 30% captisol, pH 10 and delivered to mice thorough oral gavage at 5 mg kg−1 dose. PDE3 inhibitor cilostamide stock in DMSO was diluted at 2 mg/ml in 30% captisol and delivered through intraperitoneal injection at 10 mg kg−1 dose.
Pyruvate tolerance and glycerol tolerance tests were performed in ob ob and DIO mice after overnight fasting at 1.5 g kg−1 dose using sodium pyruvate (0.2 g/ml) or 20% glycerol solution in PBS. Insulin tolerance test and glucose tolerance test were performed in ob ob mice after 8 h fasting. 2 U kg−1 insulin (Humulin R) and 0.5 g kg−1 glucose were used respectively. Blood glucose from tail bleeding was monitored by a OneTouch glucometer.
Before the test, mice were equipped with a permanent catheter in the right atrium via the jugular vein and were allowed to recover over a period of at least 3 days. After the recovery period, the mice were placed in experimental cages. All infusion experiments were performed in conscious, unrestrained mice as described previously (van Dijk et al., 2003). During the experiment, blood glucose levels were determined every 15 minutes using a Lifescan EuroFlash glucose meter. For GC-MS analysis of [U-13C] glucose, bloodspots on filter paper were collected from the tail vein every 30 minutes.
Ob/ob mice were treated for 1 week with rFGF1 (0.5 mg kg−1 every other day for one week). Steady state glucose fluxes were determined for basal and hyperinsulinemic-euglycemic clamp conditions. During the first period, mice were infused with a solution containing a tracer of [U-13C] glucose (2.5 mg/ml Cambridge Isotope Laboratories, Andover, MA) at an infusion rate of 0.54 ml/h. With respect to the final period, blood glucose levels were clamped at 20 mM. For this, the mice were infused at a constant rate of 0.135 ml/h with a mixture of insulin (44 mU/ml, Actrapid, Novo Nordisk, Bagsvaerd, Denmark), somatostatin (20 μg/ml, UCB Breda, the Netherlands), 1% BSA, and glucose (200 mg/ml from which 3% [U-13C] glucose). Additionally, a second (variable) infusion was used containing glucose (200 mg/ml from which 3% [U-13C] glucose) to adjust blood glucose levels.
Polar metabolites were extracted and analyzed using a previously reported method (Yuan et al., 2012). Briefly, ob/ob mice were sacrificed by cervical dislocation. Liver pieces (50-100 mg) were snap frozen in liquid nitrogen. 1 ml LC-MS grade 80% methanol chilled at −80° C. was added per 100 mg tissue on dry ice. Samples were homogenized by TissueLyzer and lysates were incubated on dry ice for 30 minutes and centrifuged at 20000 g for 10 minutes. Clarified supernatants were transferred to new tubes. Pellets were extracted again and supernatants were combined. After drying the supernatant under nitrogen gas, extracts were dissolved in H2O (40 μL) and 10 μL were subjected to liquid chromatography mass spectrometry (LC-MS). Polar metabolites were measured by LC-MS using a TSQ Quantiva instrument fitted with a Luna NH2 HPLC column (5.0 μm; 4.6 mm×50 mm, Phenomenex). The following LC solvents were used: buffer A, 95:5 H2O/ACN, 20 mM ammonium hydroxide, 20 mM ammonium acetate; buffer B, 100% ACN. A typical LC run was 23 minutes long with a flow rate of 0.4 ml min-1 and consisted of the following steps: 85 to 30% buffer B over 3 minutes, 30 to 2% buffer B over 9 minutes, 2% buffer B for 3 minutes, 2 to 85% buffer B over 1 minute, and 85% buffer B for 7 minutes. MS analyses were performed using electrospray ionization (ESI) in negative or positive ion mode depending on the metabolites being analyzed. Negative mode and positive mode source parameters were the following: spray voltage 3.5 kV, ion transfer tube temperature of 325° C., and a vaporizer temperature of 275° C.
Pcx activity was determined by malate dehydrogenase coupling method originally reported by Payne et al. (Payne and Morris, 1969). Briefly, mice were sacrificed by decapitation. Liver samples were rapidly frozen in liquid nitrogen within 5 seconds of excision designed to avoid loss of hepatic acetyl-CoA levels. Frozen liver samples were pulverized on dry ice and approximately 100 mg of tissues were homogenized with TissueLyzer in Pcx activity assay buffer (50 mM Tris pH8, 10 mM MgCl2, 10 mM NaHCO3). Homogenates were cleared by centrifugation at 14000 rpm for 10 minutes. Cleared supernatants were diluted in Pcx activity assay buffer to approximately 1 μg/ul. Approximately 5 μg protein was loaded onto 96-well plates and the total volume was brought up to 20 ul by Pcx activity assay buffer. Absorbance at 340 nm was monitored every 9 seconds at 37° C. immediately after the addition of 80 ul Pcx reaction buffer (50 mM Tris pH8, 10 mM MgCl2, 10 mM NaHCO3, 6.25 mM ATP, 0.125 mM NADH, 2.5 mM pyruvate, malate dehydrogenase 0.025 U/ml in Pcx activity assay buffer). Pcx activity was determined as the loss of absorbance at 340 nm over time normalized to protein concentration.
Liver samples were prepared from HFD-fed mice as described in Pcx activity assay. After determining Pcx activity from sample homogenates, the remaining supernatant was de-proteinated by perchloric acid (PCA)-KOH method using a commercial kit. De-proteinated samples were used to quantify hepatic metabolites using the commercial kits. Hepatic metabolites concentration was normalized to protein concentration and corrected for the loss of volume due to de-proteinization.
8-week old F1WT mice were sacrificed. Their gonadal adipose tissue (gWAT) was excised into small pieces (approximately 3 mm×3 mm) and maintained in saline briefly. Age-matched 8-week old F1WT and FIKO mice were anesthetized by ketamine/xylazine solution (80 mg kg−1 and 10 mg kg−1, respectively). Multiple small incisions in dorsum were made in anesthetized mice and a piece of gWAT from F1WT was placed inside each incision. Each recipient mouse received the entire gWAT from the donor mouse. Wounds were closed by wound clip. Mice were allowed to recover on heat pad. Mice were monitored, and antibiotics and pain medicine (ibuprofen) were provided post-surgery for 3 days. 2 weeks after surgery, recipient mice were sacrificed.
Isolation of adipose stromal fraction (SVF) was performed as published previously with slight modifications (Bapat et al., 2015). Inguinal adipose (iWAT) tissues were used for isolation of SVF due their ability to differentiate to mature adipocytes. Briefly, adipose tissues were dissected, washed in cold PBS and cut into small pieces in digestion buffer (100 mM HEPES, 120 mM NaCl, 50 mM KCl, 1 mM CaCl2), 1.5% fatty acids free BSA, 1 mg/ml collagenase I). Samples were dissociated in 37° C. water bath with shaking for 30-60 min with occasional monitoring to prevent over digestion. Tissue debris were filtered by 100 μm cell strainer and SVF was collected by centrifugation at 500 g for 5 minutes. Cell pellet was washed once by PBS and filtered with 40 μm cell strainer. Red blood cells were lysed with Red blood cell lysis buffer (BioLegend) according to manufacturer's instructions. The remaining cells were re-suspended and cultured in DMEM/F12 with 20% FBS (GemCell, 100-500) at 37° C., 5% CO2.
Differentiation of pre-adipocytes were based on previously published methods (Bunnell et al., 2008). Briefly, 3T3-L1 pre-adipocytes were grown to full confluence in DMEM, 10% FBS (GemCell, 100-500), 10 mM HEPES and antibiotic-antimycotic (full growth media). 2 days later, differentiation was induced by replacing the media with 1 μM dexamethasone, 1 μM rosiglitazone, 500 μM IBMX and 5 μg/ml insulin in full growth media 2 days later after reaching full confluence. Two days later media was replaced by differentiation induction media with 1 μM rosiglitazone and 5 μg/ml insulin for 4 days with media change after 2 days. Cells were kept in maintenance medium (full growth media with 5 μg/ml insulin) for 4 more days for full differentiation. For differentiation of mouse SVF derived adipocytes, the protocol was same except DMEM/F12 (10565018, ThermoFisher) was used as base media and initial differentiation induction media was kept for 3 days. For differentiation of human subcutaneous adipocytes, protocol and media from the manufacturer (C-12735, C-39437, Promocell) were used. Dexamethasone was omitted from the media in the last 3 days before performing experiments. Both mouse and human SVF derived adipocytes were differentiated in collagen coated plates (A1142802, ThermoFisher).
For in vitro lipolysis assay, cells were washed with PBS and media were changed to full growth media 1 day before experiment. Cells were serum starved for 2 h and placed in KRBH buffer (30 mM HEPES, 120 mM NaCl, 4 mM KH2PO4, 1 mM MgSO4, 0.75 mM CaCl2), and 10 mM NaHCO3) with 2% fatty acid free BSA and 5 mM glucose. FGF1 was added 10 min before induction of lipolysis with 100 nM isoprotenol (ISO). Inhibitors were added 30 min prior to FGF1 treatment unless otherwise noted. Media were collected and cells were lysed in protein extraction buffer. FFAs were measured in the media using a commercial kit (Wako-NEFAHR2) and normalized to protein concentration. For ex vivo lipolysis, the assay was modified from a published protocol (Funicello et al., 2007). Briefly, approximately 0.1 gram of adipose tissue was excised, weighed and kept in cold PBS until treatment and control tissues were collected. Tissues were cut into small pieces and incubated in same KRBH buffer as described above for 4 h. FFAs were measured and normalized by the explant weights.
Tissues were lysed in cold lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% NP40, 0.5% NaDoc, 0.1% SDS, 5% glycerol, 1 mM EDTA, protease and phosphatase inhibitors (complete and PhosSTOP (Roche)) by homogenization by bead-beater for 30 s. Samples were cleared for 10 min at 18,000 g at 4° C. and the middle clear phase was transferred to new tubes. A second 30 min centrifugation was performed and the middle clear phase was transferred to new tubes. Five ul protein extract was used for BCA assay to determine protein concentration. Samples were boiled in Laemmli buffer. SDS-PAGE and blotting were performed using gradient gels and Trans-Blot Turbo Transfer System (Bio-Rad). Antibodies used were PDE4D (12918-1-AP, Proteintech), pHSL-660 (4126S, Cell signaling), HSL (4107S, Cell Signaling), tubulin (CP06, Millipore Sigma), and GFP (A01388, GenScript). PDE4D3-pS44 phospho-specific antibody was generated using PDE4D3-S44 FRRHpSWISFDVDNGTSAGR peptide (SEQ ID NO: 20) (Mackenzie et al., 2002) by Pocono Rabbit Farm, using 70-day rabbit antibody production protocol and purified via affinity purification. Primary antibodies were incubated for 2 h at room temperature (RT) or overnight (ON) at 4° C. Blots were developed using SuperSignal West Pico PLUS Chemiluminescent Substrate (ThermoFisher Scientific) and imaged with BioRad GelDoc system.
Virapower gateway adenovirus expression kit was used to clone ORFs of various isoforms of mouse PDE4D (XM_006517645.4, XM_030247262.1 and XM_006517647.3) to adenoviral vectors according to the manufacturer's protocol. Vectors were transfected into 293A cell line using Fugene transfection reagent (PRE2692, Promega). Crude adenovirus stock was used to infect new 293A cells for large-scale adenovirus production. Transduced 293A cells were harvested when cytopathic effect was apparent 2-3 days after inoculation with crude adenovirus stock. Virus were purified via adenovirus standard purification kit (3054, Virapur). For adipose specific expression of target proteins by AAV mediated transduction (adAAV), the vector was designed by using a human adiponectin promoter/enhancer (SEQ ID NO: 27) based on O'Neil et al. (O'Neill et al., 2014) and 4× repeat miRNA target for miR 122T (SEQ ID NO: 28) (Qiao et al., 2011) after cloned ORF to further prevent liver expression. adAAV backbone was synthesized by Vector Builder. ORFs of PDE4D3, GFP, mCherry, Perilipin-GFP, HSL-mCherry were cloned to the vector with standard restriction digestion cloning. cDNA from gonadal adipose tissue was used to amplify the ORFs with the primers listed at Table 1. Stabl3 cells were used for vector amplification and Hi Pure Purelink Expi Plasmid Giga Prep (ThermoFisher) was used for AAV vector purification. Large scale AAV8 production and calculation of the titer were performed by Salk Institute Gene Transfer, Targeting, and Therapeutics Core (GT3). Mutagenesis experiments were performed by using QuickChange XL Site-Directed Mutagenesis Kit (200517, Agilent). Primer sequences are listed at Table 1.
PDE activities were measured by using [3H]-labelled cAMP as described previously with slight modifications (Rybalkin et al., 2013). Briefly, 10 μg of protein extract was incubated in assay buffer (20 mM Tris pH 7.4, 0.8 mM EGTA pH 8, 0.2 mg/ml BSA, 15 mM magnesium acetate, 1 μM cAMP, 50000 cpm [3H]-cAMP) in 250 ul volume at 30° C. for 15 min. Reaction was stopped by adding 125 ul, 0.25M HCl and neutralized by adding 125 ul 0.25M NaOH and final 100 mM Tris-HCl pH 7.4. Five ul Crotalus atrox venom (BML-KI307) was used for dephosphorylation of [3H]-5-AMP at 30° C. for 30 min. [3H]-adenosine product was separated from [3H]-cAMP substrate by ion-exchange chromatography (DEAE-Sephadex A-25; GE Healthcare) and quantified by scintillation counting.
3T3-L1 cells were transduced with viral particles (10+-106GC/cell) in 24 well plates. Media was replaced after overnight incubation. 3-4 days after AAV infection, cells were treated as described for lipolysis assay. For cAMP analysis by biosensors, Downward Green cADDis cAMP Sensor (D0200G) and control mNeon Green (F0500G) produced in BacMam system was purchased from Montana Molecular. Brightfield and fluorescence images were taken every 10-20 min in I IncuCyte® Live-cell analysis system (Sartorius) and images were analyzed by IncuCyte® Analysis Software. High-resolution live cell imaging was performed with LSM 880 Airyscan microscope at 40× objective.
Animal experiments were performed on multiple cohorts. In vitro experiments were performed at least 3 times. The randomized block design was used for all animal experiments. The age, sex, body weight and cage effect were identified as blocking factors. Therefore, all animal experiments were carried out on age-matched animals of the same sex. Body weights were measured before assigning treatment groups. Cage effect was controlled in pharmacological treatment studies by assigning animals to the placebo or treatment group from the same cage.
Pre-determined sample exclusion criterion was established for technical failures. Unless otherwise noted, statistical significance was calculated by unpaired, two-tailed student's t test. In time series data, two-way ANOVA was performed. Data are presented as mean±SEM.
To validate that FGF1-induced glucose lowering is dependent on FGFR1 expression in adipose tissue, FGFR1 was selectively deleted in mature adipocytes (Fgfr1 f1/f1 crossed to adiponectin-CRE, adR1KO mice). FGF1 rapidly decreased blood glucose levels in diet-induced obese (DIO) wild type mice (0.5 mg/kg FGF1 s.c., adR1WT) but failed to affect adR1KO mice, consistent with previous findings (Suh et al., 2014) (
To determine whether exogenous FGF1 can similarly affect adipose lipolysis in vivo, DIO adR1WT and adR1KO mice were fasted overnight to minimize compensatory changes in insulin prior to injection with FGF1 (0.5 mg/kg s.c.). FGF1 reduced serum FFA levels in adR1WT mice by ˜30%, but failed to affect adR1KO mice (
The above findings implicate FGF1-FGFR1 signaling as a novel pathway regulating adipose lipolysis. This regulation appears specific to white adipose depots, as FGF1 did not affect lipolysis in brown adipose tissue (
Insulin regulates blood glucose levels in part by suppressing lipolysis and thereby HGP, and dysregulated HGP contributes to hyperglycemia in insulin resistance (Boden et al., 2017; Lin and Accili, 2011; Lombardo and Menahan, 1979; Turner et al., 2005). To determine whether the suppression of lipolysis by FGF1 acutely reduced HGP, the ability of FGF1 to affect gluconeogenic substrate utilization was measured. oblob mice pretreated with FGF1 had a markedly reduced ability to synthesize glucose from pyruvate (pyruvate tolerance test; PTT), while no differences were seen when glycerol was the exogenous substrate (glycerol tolerance test; Glycerol TT) (
To test the relevance of these findings to glucose homeostasis, a hyperinsulinemic clamp was performed on ob/ob mice after short-term serial FGF1 administration (0.5 mg/kg every other day for a week). This limited treatment regimen resulted in a ˜25% reduction in basal endogenous glucose production (EGP) (
Insulin suppresses lipolysis via the PI3K-dependent activation of PDE3B (DiPilato et al., 2015a; Rahn et al., 1994). As FGFR1 activation can also signal via the PI3K pathway, it was investigated whether the anti-lipolytic effects of FGF1 were affected by the PI3K inhibitor wortmannin. Paralleling insulin signaling, wortmannin abrogated the FGF1-induced reduction in FFA release in 3T3-L1 adipocytes (
Given the above findings, it was posited that FGF1-PDE4 signaling regulated HSL phosphorylation. Indeed, in both basal and isoproterenol-stimulated cells, the ability of FGF1 to suppress HSL phosphorylation was lost in the presence of the PDE4 inhibitor roflumilast (
In combination, these findings suggested a model in which FGF1-FGFR1 activation of PDE4 attenuates cAMP/PKA phosphorylation of HSL and its subsequent association with perilipin on the lipid droplet surface. Based on previous studies linking PDE4D with adipose lipolysis, it was explored whether overexpression of PDE4D is sufficient to recapitulate the ability of FGF1 to suppress lipolysis (Jang et al., 2019). Indeed, overexpression of 3 PDE4D isoforms dose-dependently suppressed lipolysis in 3T3-L1 adipocytes, with the PDE4D3 isoform showing the highest efficacy (
The finding that FGF1-induced suppression of lipolysis is dependent on PDE4 raised the possibility that this pathway contributes to glucose homeostasis in insulin-resistant mice. To explore this possibility, ad lib fed DIO mice were treated with the PDE4 inhibitor roflumilast. In these mice, PDE4 inhibition transiently increased blood glucose, serum FFA and insulin levels (
The activities of phosphodiesterases are regulated by multiple phosphorylation events that integrate different signaling pathways (Mika and Conti, 2016). To explore whether FGF1 signaling induces PDE4D phosphorylation, isoproterenol-stimulated 3T3-L1 adipocytes were treated with FGF1. PDE4D was phosphorylated upon isoproterenol treatment, as evidenced by its decreased mobility in an SDS-PAGE gel (
PDE4D proteins are phosphorylated by PKA at a conserved S85 site in the upstream conserved region 1 (S54 in humans and rats) that is thought to be necessary for activation, as well as at S44 (S13 in humans and rats), a PDE4D3-specific site in the N-terminus that does not affect PDE activity in vitro (
The above data indicate that the specific phosphorylation of PDE4D at S44 is required for the anti-lipolytic activity of FGF1/PDE4D pathway. To confirm the in vivo relevance of this finding, ob/ob mice were injected with adAAV-GFP, adAAV-PDE4D3 or adAAV-PDE4D3 S44A (adipose tissue-specific expression was confirmed by Western blot;
FGF1 was identified as a fed-state adipokine whose expression is increased in response to high fat diet feeding (Jonker et al, 2012). In order to associate endogenous FGF1 signaling with S44 phosphorylation, gWAT depots were collected from chow and HFD fed mice under overnight fasted and refed conditions. Re-feeding approximately doubled the pS44 levels in both chow and HFD-fed mice. Additionally, HFD markedly reduced S44 phosphorylation in both the fasted and fed states, suggestive of a role for PDE4D in insulin-resistant hyperlipidemia (
In view of the many possible aspects to which the principles of the disclosed subject matter may be applied, it should be recognized that the illustrated aspects are only examples of the disclosure and should not be taken as limiting the scope of the disclosure. Rather, the scope of the disclosure is defined by the following claims. We therefore claim all that comes within the scope and spirit of these claims.
This application claims the benefit of U.S. Provisional Application No. 63/294,563, filed Dec. 29, 2021, which is herein incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/082455 | 12/28/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63294563 | Dec 2021 | US |
