Use of uridine diphosphate glucose in treatment of abnormal liver glucolipid metabolism

Abstract

A use of uridine diphosphate glucose (UDPG) in treatment of abnormal liver glucolipid metabolism. Specifically, a use of UDPG or a pharmaceutical composition comprising same in the preparation of a drug for preventing and / or treating abnormal liver glucolipid metabolism, wherein the abnormal liver glucolipid metabolism is preferably nonalcoholic fatty liver disease, dyslipidemia and related cardiovascular diseases. Treatment using UDPG can reduce lipid synthesis and lipid synthesis related gene expression in the liver.

Description

Use of uridine diphosphate glucose in treating abnormal liver glucose and lipid metabolism Technical Field

[0001] The present invention relates to the field of biomedicine, and more particularly to the use of uridine diphosphate glucose in treating abnormal liver glucose and lipid metabolism. Background Art

[0002] Glucose and lipid disorders (GLMDs) refer to a comprehensive group of diseases characterized by impaired glucose and lipid metabolism, encompassing a range of pathological conditions, including obesity, type 2 diabetes mellitus (T2DM), hypertension, dyslipidemia, non-alcoholic fatty liver disease (NAFLD), and atherosclerosis. With the changing lifestyles and improved living standards of modern society, GLMDs have become a major chronic disease contributing to global public and clinical problems. These conditions are closely interrelated, often presenting with multiple comorbidities, making treatment challenging and a recognized threat to human health. Research has shown that, while the pathogenesis of GLMDs remains incompletely understood, it is well established that various GLMDs share a common core risk factor: impaired glucose and lipid metabolism. While the normal function of systemic glucose and lipid metabolism depends on a high degree of coordination among various tissues and organs, the liver, as a major metabolic control center responsible for maintaining energy and nutrient homeostasis under changing physiological conditions, is a central hub of glucose and lipid metabolism and plays a significant role in the development and progression of various GLMDs.

[0003] NAFLD is one of the main causes of abnormal liver glucose and lipid metabolism. Data indicate that approximately 23% of patients worldwide suffer from NAFLD, making it a major contributor to the global prevalence of liver disease. Data indicate that NAFLD is even more severe in developing countries. For example, in my country, the prevalence of NAFLD is as high as 29.3%. We urgently need to address the severe challenges posed by the widespread prevalence of NAFLD. However, to date, there are no FDA-approved drugs for the treatment of NAFLD, and the regulatory mechanisms of glucose and lipid metabolism in the liver remain incompletely understood. Therefore, in-depth research into the pathogenesis of NAFLD is crucial for the development of new drugs.

[0004] On the other hand, abnormal liver lipid metabolism can lead to elevated blood lipids, resulting in high cholesterol and / or high triglycerides. It is well known that elevated blood lipids are directly associated with an increased risk of atherosclerotic cardiovascular disease.

[0005] Research has shown that the liver is a central organ responsible for the synthesis, metabolism, storage, and redistribution of glucose and lipids. Within the liver, glucose and lipid metabolism are closely intertwined in several ways. The body's inability to degrade excess lipids produced in the liver leads to excessive lipid deposition and the accumulation of harmful lipids, which in turn causes liver damage and is one of the main causes of NAFLD. Therefore, regulating lipid production in the liver and inhibiting excessive lipid deposition and accumulation of harmful lipids in the liver opens up new avenues for the prevention and treatment of NAFLD.

[0006] Summary of the Invention

[0007] The present invention is proposed to solve the problem that there is currently no standard treatment for NAFLD and no specific medicine for NAFLD.

[0008] The present inventors unexpectedly discovered that uridine diphosphate glucose (UDPG) can effectively inhibit lipid synthesis in the liver, significantly reduce the levels of triglycerides and cholesterol in the liver and / or serum, and inhibit fat accumulation in the liver.

[0009] Thus, in a first aspect, the present invention provides the use of uridine diphosphate glucose (UDPG) in the preparation of a medicament for preventing and / or treating a disease associated with abnormal liver glucose and lipid metabolism in a subject.

[0010] In another aspect, the present invention provides a use of a pharmaceutical composition comprising uridine diphosphate glucose in the preparation of a medicament for preventing and / or treating a disease associated with abnormal liver glucose and lipid metabolism in a subject, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0011] The diseases associated with abnormal liver glucose and lipid metabolism include, but are not limited to, insulin resistance, metabolic syndrome, obesity, type II diabetes, dyslipidemia, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), coronary heart disease, hypertension and atherosclerosis.

[0012] In a preferred embodiment, the abnormal liver glucose and lipid metabolism is NAFLD. In a preferred embodiment, the NAFLD is early-stage NAFLD. In a preferred embodiment, the NAFLD is NAFLD induced by a high-fat diet.

[0013] In specific embodiments, the dyslipidemia is hyperlipidemia, including but not limited to hypercholesterolemia and / or hypertriglyceridemia.

[0014] According to some embodiments of the present invention, the subject is a mammal, preferably a human.

[0015] According to some embodiments of the present invention, the medicament is formulated as a dosage form for oral or parenteral administration, preferably formulated as a dosage form for oral, intradermal, subcutaneous, intraperitoneal, intrahepatic or intravenous administration.

[0016] According to some embodiments of the present invention, the UDP-glucose reduces lipid synthesis in the liver of the subject, preferably, the UDP-glucose reduces the synthesis of triglycerides and / or cholesterol in the liver.

[0017] According to some embodiments of the invention, the UDP-glucose reduces the level of triglycerides and / or total cholesterol in the serum of the subject.

[0018] According to some embodiments of the present invention, the subject suffers from or is suspected of suffering from or has a risk of suffering from abnormal liver glucose and lipid metabolism.

[0019] In another aspect, the present invention provides a method for preventing and / or treating a disease associated with abnormal liver glucose and lipid metabolism in a subject, the method comprising administering a therapeutically effective amount of uridine diphosphate glucose to the subject.

[0020] In a specific embodiment, the present invention provides a method for preventing and / or treating a disease associated with abnormal liver glucose and lipid metabolism in a subject, the method comprising administering a therapeutically effective amount of uridine diphosphate glucose to the subject; the disease associated with abnormal liver glucose and lipid metabolism includes but is not limited to insulin resistance, metabolic syndrome, obesity, type II diabetes, dyslipidemia, NAFLD, NASH, coronary heart disease, hypertension, and atherosclerosis.

[0021] In a specific embodiment, a therapeutically effective amount of UDP-glucose is administered to the subject orally or parenterally, preferably orally, intradermally, subcutaneously, intraperitoneally, intrahepatically or intravenously.

[0022] In another aspect, the present invention provides an in vitro method for reducing lipid synthesis in hepatocytes or liver organoids, comprising the step of contacting the hepatocytes or liver organoids with uridine diphosphate glucose in vitro.

[0023] In a specific embodiment, the present invention provides an in vitro method for reducing lipid synthesis in hepatocytes or liver organoids, the method comprising the steps of:

[0024] 1) Culturing hepatocytes or liver organoids in vitro under conditions suitable for their growth; and

[0025] 2) contacting the hepatocytes or liver organoids cultured in vitro in step 1) with uridine diphosphate glucose in vitro.

[0026] After extensive experimental research, the inventors discovered that UDPG can effectively inhibit lipid synthesis in the liver and is a novel therapeutic strategy for NAFLD. Glycogen production in hepatocytes is shunted to the Golgi apparatus by transporting the intermediate metabolite UDPG via solute transporter 35 (SLC35). UDPG inhibits the activity of site 1 protease (S1P) in the Golgi apparatus, inhibiting S1P-mediated cleavage of sterol regulatory element binding protein (SREBP), thereby inhibiting lipid synthesis in the liver. In vivo studies have found that compared with the control group, UDPG treatment can reduce liver steatosis in a fatty liver mouse model and inhibit liver lipid production in liver organoids and NAFLD patients.

[0027] Compared with other drugs in clinical trials, UDPG has the following obvious advantages: (1) UDPG exists directly in the human peripheral blood and can be effectively administered by intravenous injection; (2) UDPG itself can participate in the process of converting monosaccharides into glucose in the human body and is patient-friendly; (3) UDPG can be taken up by liver cells, ensuring its feasibility in regulating liver lipid production; (4) UDPG may promote glycogen metabolism, which scavenges reactive oxygen species (ROS) by regulating the pentose phosphate pathway. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG1A and FIG1B show that UDPG can reduce lipid synthesis and the expression of lipid synthesis-related genes in mouse hepatocytes.

[0029] Figures 2A to 2F show that UDPG can reduce the body weight of mice with fatty liver and reduce the lipid and cholesterol synthesis in the liver and serum.

[0030] Figures 3A to 3C show that the level of UDPG in hepatocytes of NAFLD patients is low, and UDPG can reduce lipid synthesis and the expression of lipid synthesis-related genes in adult hepatocytes.

[0031] Figures 4A to 4C show that UDPG can reduce lipid synthesis in human liver organoids. DETAILED DESCRIPTION

[0032] To prevent and / or treat diseases associated with abnormal liver glucose and lipid metabolism, the drug or pharmaceutical composition of the present invention can be used alone or in combination with surgical intervention, endocrine therapy, drug therapy, and the use of biological response modifiers.

[0033] In some specific embodiments, the medicament or pharmaceutical composition of the present invention comprises uridine diphosphate glucose (UDPG) as an active ingredient, and optionally comprises a pharmaceutically acceptable carrier or excipient.

[0034] Uridine diphosphate glucose, or UDPG, is a nucleotide sugar widely distributed within microbial, animal, and plant cells. It serves as a glucose donor in the biosynthesis of various glycosides, oligosaccharides, and polysaccharides. UDPG also plays a central role in carbohydrate metabolism, serving as an important intermediate in the interconversion of monosaccharides and the production of furfural acid.

[0035] In other specific embodiments, UDPG is used in combination with one or more other treatment methods or therapeutic agents, preferably chemotherapy or targeted therapy, and preferably another agent for preventing and / or treating abnormal liver glucose and lipid metabolism.

[0036] The medicament or pharmaceutical composition of the present invention can be conveniently presented in unit dosage form. The medicament or pharmaceutical composition of the present invention can be formulated into any suitable dosage form, such as, but not limited to, an injection, tablet, capsule, gel, etc. The medicament or pharmaceutical composition of the present invention can also be formulated into a suspension in an aqueous, non-aqueous, or mixed medium. The medicament or pharmaceutical composition of the present invention includes, but is not limited to, solutions, emulsions, foams, and liposome-containing formulations. The pharmaceutical composition of the present invention may include one or more penetration enhancers, carriers, and excipients.

[0037] In some embodiments, the diseases that can be intervened by the drugs or pharmaceutical compositions of the present invention are selected from any one of the following or a combination thereof: insulin resistance, metabolic syndrome, obesity, type II diabetes, dyslipidemia, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, coronary heart disease, hypertension, and atherosclerosis.

[0038] In some specific embodiments, the medicament or pharmaceutical composition of the present invention is used to prevent and / or treat NAFLD.

[0039] As used herein, the term "non-alcoholic fatty liver disease (NAFLD)" is a common liver disease, a liver manifestation of metabolic syndrome, and a concentrated reflection of related diseases such as obesity, insulin resistance, type 2 diabetes, hypertension, and hyperlipidemia.

[0040] As used herein, the term "dyslipidemia" refers to a common disorder characterized by abnormal lipoprotein metabolism in the human body, primarily manifested by elevated serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), and / or decreased high-density lipoprotein cholesterol (HDL-C). Dyslipidemia is a key factor contributing to atherosclerosis and an independent risk factor for coronary heart disease and ischemic stroke.

[0041] The term "treat" refers to eliminating the disease, arresting the progression of the disease, slowing the progression of the disease, reducing the duration of one or more symptoms associated with the disease, improving or reversing at least one measurable parameter associated with the disease, or increasing the survival of a subject suffering from the disease.

[0042] In some embodiments, UDPG or a pharmaceutical composition containing the same is administered to a subject in a therapeutically effective amount.

[0043] The term "therapeutically effective amount" may refer to the amount of an active ingredient or pharmaceutical composition that induces an animal or human to exhibit a biological or medical response considered by a researcher, veterinarian, doctor or other clinician, and such amount may include the amount of an active ingredient or pharmaceutical composition that is used to induce relief of the disease or condition to be treated. It will be apparent to those skilled in the art that the therapeutically effective dose and number of administrations of the active ingredient of the present invention may vary depending on the desired effect. The term "subject" refers to an animal, preferably a mammal. According to a specific embodiment, the subject is a mammal, including, for example, camels, donkeys, zebras, cattle, pigs, horses, goats, sheep, cats, dogs, rats, rabbits, guinea pigs, mice, primates (e.g., humans). In a specific embodiment, the subject is a human. In a specific embodiment, the subject is a human who is susceptible to, suspected of having, or has suffered from abnormal liver glucose and lipid metabolism, preferably NAFLD. The term "administer" may refer to providing a predetermined substance to a subject by any appropriate method.

[0044] The administration amount or intake amount can be administered in various administration doses and methods by distributing the composition according to the subject's weight, age, sex, health condition, diet, administration time, administration method, excretion rate and disease severity, for example, once a day or multiple times a day.

[0045] In the present invention, the administration route of the pharmaceutical composition of the present invention can be administered by any general route as long as it can reach the target tissue. It can be administered orally, intraperitoneally, intravenously, intramuscularly, subcutaneously, endothelially, intranasally, intrapulmonary, rectal, intracavitary, intraperitoneally and intrathecally, but is not limited thereto. In a preferred embodiment, the drug is administered parenterally, preferably by intravenous injection.

[0046] Example

[0047] Experimental Materials Male wild-type C57BL / 6 mice were purchased from the Medical Laboratory Animal Center of the Chinese Academy of Medical Sciences (Beijing, China). These animals were maintained under sterile conditions in the animal facility of the Chinese Academy of Medical Sciences. All studies involving mice were approved by the Animal Care and Use Committee of the Chinese Academy of Medical Sciences (ACUC-A02-2020-009).

[0048] All research protocols involving human liver tissue samples were approved by the First Affiliated Hospital of Zhengzhou University. All protocols adhered to the Declaration of Helsinki and the Declaration of the World Medical Association, and informed consent was signed by all individuals or their family members.

[0049] Example 1. UDPG can reduce lipid synthesis in mouse hepatocytes.

[0050] 1. Experimental Procedure

[0051] Primary hepatocytes were isolated from mice by a two-step collagenase perfusion method.

[0052] 1.6×10 5 Cells were plated into 6-well plates. After cell attachment, hepatocytes in the experimental group were treated with 100 μM and 200 μM UDPG, while hepatocytes in the control group were treated with the same concentration of G1P. After 48 hours of treatment, the culture medium was discarded and the cells were washed twice with PBS. The PBS was completely discarded after the final wash, and the cells were scraped and TG levels were measured using a cuvette assay.

[0053] Primary hepatocytes were isolated from mice by a two-step collagenase perfusion method, with 1.6 × 10 5 Cells were plated into 6-well plates. After cell attachment, hepatocytes were treated with 100 μM and 200 μM UDPG for 48 hours. The culture medium was discarded and the cells were washed twice with PBS, with the PBS completely discarded after the final wash. 1 mL of TRIZOL was added to each well, and cellular RNA was extracted and reverse transcribed. The expression of lipid synthesis-related genes Acaca, Fasn, and Scd1 was analyzed by qPCR.

[0054] 2. Experimental Results

[0055] Compared with the control group, treatment of hepatocytes with 100 μM and 200 μM UDPG reduced TG levels, indicating decreased lipid synthesis (Figure 1A). qPCR analysis showed that UDPG treatment could reduce the expression of lipid synthesis-related genes Acaca, Fasn, and Scd1 in hepatocytes (Figure 1B).

[0056] Example 2. UDPG can reduce the body weight of mice with fatty liver and reduce the synthesis of lipids and cholesterol in the liver and serum.

[0057] 1. Experimental Procedure

[0058] C57BL / 6 mice were divided into three groups (n=6 per group): one group was fed a normal diet (ND) and two groups were fed a high-fat diet (HFD). After 8 weeks of feeding, one group of HFD mice received an intraperitoneal injection of UDPG (8 mg / kg) (HFD+UDPG group), while the other groups of HFD mice and ND mice received an intraperitoneal injection of an equal volume of PBS solution (ND+vehicle group and HFD+vehicle group). The mice were weighed daily. After 8 weeks of injection, the mice were sacrificed, and liver tissue, blood, and serum were collected. A portion of liver tissue and serum was obtained, and triglyceride and TC levels in the liver tissue and blood were measured using a colorimetric method. A portion of liver tissue was fixed in 4% paraformaldehyde for 48 hours, embedded in paraffin, and pathological sections were prepared. Hematoxylin and eosin (H&E) and oil red (Oli Red O) staining were performed, and the sections were observed under a microscope.

[0059] 2. Experimental Results

[0060] Mice in the HFD+UDPG group weighed less than those in the HFD+vehicle group (Figure 2A). Triglyceride and TC levels in the liver and serum were significantly lower than those in the HFD+vehicle group (Figures 2B-2E), indicating lower lipid and cholesterol synthesis. The livers of mice in the HFD+vehicle group showed vesicular steatosis and prominent lipid droplets, whereas the livers of mice in the HFD+UDPG group showed no significant degeneration, and the lipid droplets were significantly smaller than those in the HFD+vehicle group (Figure 2F).

[0061] Example 3. UDPG can inhibit lipid synthesis in hepatocytes of NAFLD patients.

[0062] 1. Experimental Procedure

[0063] Primary hepatocytes were isolated from liver tissue of NAFLD patients by two-step collagenase perfusion method, and cell suspension was collected and cultured at a rate of 1.6 × 10 5 Cells were plated into 6-well plates. After the cells adhered, they were washed twice with PBS. The PBS was completely discarded after the last wash. The liver cells were scraped and the UDPG level in the patient's liver tissue was measured.

[0064] Primary hepatocytes were isolated from adult liver tissue by a two-step collagenase perfusion method, and the cell suspension was collected and cultured at a rate of 1.6 × 10 5Cells were plated into 6-well plates. After the cells adhered, the experimental group was pretreated with 200 μM UDPG, while the control group was treated with an equal amount of PBS. The cells were then treated with 400 μM free fatty acids (FFA) for 48 hours. The culture medium was discarded, and the cells were washed twice with PBS. The PBS was completely discarded after the last wash. The hepatocytes were scraped and a portion of the cells was used to measure the TG levels of the two groups using a cuvette method. A portion of the cells was added with TRIZOL, and the cellular RNA was extracted and reverse transcribed. The expression of the lipid synthesis genes Acaca, Fasn, and Scd1 was analyzed by qPCR.

[0065] 2. Experimental Results

[0066] UDPG levels in liver tissue from patients with NAFLD were much lower than those in normal liver tissue (Figure 3A). When FFA was used to treat adult hepatocytes, TG levels in cells pretreated with UDPG were significantly lower than those in the control group (Figure 3B), indicating lower lipid synthesis. The expression of lipid synthesis-related genes Acaca, Fasn, and Scd1 was also significantly lower than that in the control group (Figure 3C).

[0067] Example 4. UDPG can reduce lipid synthesis in liver organoids.

[0068] 1. Experimental Procedure

[0069] Primary hepatocytes were isolated from adult liver tissue by a two-step collagenase perfusion method. The cell suspension was collected and mixed with the prepared Matrigel gel at a ratio of 1:3. The cell density was adjusted according to the cell counting results to achieve 1×10 cells per 12-well plate. 4 Hepatocytes were added to each well. 20 μL of the mixture at different concentrations was then dropped onto the center of the plate to form a spheroid. The mixture was allowed to stand for a moment until it solidified slightly, and the plate was carefully inverted and placed in the cell incubator. After 1 hour, 500 μL of culture medium was added to each well. The growth and morphological changes of the liver organoids were observed daily, and the culture medium was replaced every 2-3 days.

[0070] After 2-3 days of growth, the liver organoids were pretreated with 200 μM UDPG in the experimental group and an equal volume of PBS in the control group. The cells were then treated with 400 μM oleic acid (OA) for 48 hours. The culture medium was discarded and the cells were washed twice with PBS, with the PBS completely discarded after the final wash.

[0071] For TG measurement, 200 μL of cell recovery solution was added to each well. The Matrigel in the well was broken up by pipetting and then aspirated. The mixture was collected into an EP tube and incubated on ice until the Matrigel was completely melted. The mixture was centrifuged at 800 rpm for 5 minutes at 4°C. The supernatant was discarded, and 500 μL of trypsin was added for digestion. The mixture was incubated at 37°C and observed under a microscope until the colonies were dispersed into single cells. The cells were then collected by centrifugation at 800 rpm for 5 minutes.

[0072] For morphological observation, liver organoids with the most complete morphology possible were digested from the matrix gel and collected into EP tubes. After fixation with 4% paraformaldehyde (PFA) on ice for 1 hour, they were washed with cold PBS and centrifuged at 4°C, 1500rpm for 5 minutes, and repeated 2-3 times. The cells were then resuspended with 4% PFA and fixed overnight at 4°C. After fixation, centrifugation was performed and the 4% PFA was discarded. 2% low-melting-point agarose was prepared using sterile PBS and heated in a microwave oven until completely melted. The liver organoids were resuspended in the still warm agarose liquid and immediately added to a ring mold placed on ice. They were placed on ice for 1 hour until the agarose was completely solidified. The solidified agarose blocks can be subsequently embedded in paraffin, sectioned, and stained with boron dipyrrolidone (Bodipy).

[0073] 2. Experimental Results

[0074] Primary hepatocytes can rapidly expand in a 3D culture environment. After 3 days of induction culture, they can expand from single cells to clones with a diameter of 100 μm, and form organoids with cystic structures of 300 μm on the 7th day (Figure 4A). When organoids were treated with oleic acid, the TG level in organoids pretreated with UDPG was significantly lower than that in the control group (Figure 4B), indicating lower lipid synthesis.

[0075] Obvious lipid particles appeared in the Bodipy-stained sections of organoids in the control group, while there were significantly fewer lipid particles in the UDPG group (Figure 4C).

[0076] The results of the above examples show that:

[0077] UDPG can effectively inhibit lipid synthesis in the liver and is a novel treatment strategy for NAFLD;

[0078] In vivo studies have found that compared with the blank control group, UDPG treatment can significantly reduce lipid synthesis in the liver of animal models and alleviate the degree of hepatic steatosis;

[0079] Compared with other drugs in clinical trials, UDPG exists directly in the human peripheral blood and can be effectively administered by intravenous injection; it can also be taken up by liver cells, ensuring its feasibility in regulating liver lipid production. It is highly safe and has no toxic side effects, and can be used directly in the human body.

Claims

1. Use of UDP-glucose in the preparation of a medicament for preventing and / or treating a disease associated with abnormal liver glucose and lipid metabolism in a subject.

2. Use of a pharmaceutical composition comprising UDP-glucose in the preparation of a medicament for preventing and / or treating a disease associated with abnormal liver glucose and lipid metabolism in a subject, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

3. The use according to claim 1 or 2, wherein the disease associated with abnormal liver glucose and lipid metabolism is selected from the group consisting of insulin resistance, metabolic syndrome, obesity, type II diabetes, dyslipidemia, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, coronary heart disease, hypertension and atherosclerosis.

4. The use according to claim 1 or 2, wherein the dyslipidemia is hyperlipidemia, preferably hypercholesterolemia and / or hypertriglyceridemia.

5. The use according to claim 1 or 2, wherein the subject is a mammal, preferably a human.

6. The use according to claim 1 or 2, wherein the medicament is formulated into a dosage form for oral or parenteral administration, preferably formulated into a dosage form for oral, intradermal, subcutaneous, intraperitoneal, intrahepatic or intravenous administration.

7. The use according to claim 1 or 2, wherein the UDP-glucose reduces lipid synthesis in the subject's liver, preferably, the UDP-glucose reduces the synthesis of triglycerides and / or cholesterol in the liver.

8. The method according to claim 1 or 2, wherein the UDP-glucose reduces the content of triglycerides and / or total cholesterol in the serum of a subject.

9. The use according to claim 1 or 2, wherein the subject suffers from or is suspected of suffering from or has a risk of suffering from abnormal liver glucose and lipid metabolism.

10. An in vitro method for reducing lipid synthesis in hepatocytes or liver organoids, the method comprising the step of contacting the hepatocytes or liver organoids with uridine diphosphate glucose in vitro.

Images