In vitro hepatic glucose production assay

Abstract

Provided herein are hepatic glucose production (HGP) assay methods which measure changes in total HGP, HGP due to glycogenolysis, and / or HGP due to gluconeogenesis, in response to a compound. Also provided herein are HGP assay methods which identify hepatic cells that are responsive to compounds by measuring changes in total HGP, HGP due to glycogenolysis, and / or HGP due to gluconeogenesis. Further provided herein are kits of reagents suitable for preparation of samples for in vitro measurement of HGP.

Description

IN VITRO HEPATIC GLUCOSE PRODUCTION ASSAYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of Singapore provisional application no. 10202401296W, filed on 03 May 2024, the contents of it being hereby incorporated by reference in its entirety for all purposes.FIELD OF THE INVENTION

[0002] The present invention relates to the field of biotechnology. In particular, the present invention relates to assays and methods of screening of hepatic glucose production in hepatocytes.BACKGROUND OF THE INVENTION

[0003] High circulating glucose is a key attribute of diabetes and is contributed to by ineffective glucose uptake by musclc / fat tissue, ineffective insulin secretion from the pancreatic beta cells, or defective hepatic glucose regulation. Researchers have been actively pursuing insulin resistance in muscle / fat and defective beta cells, and liver-centric researchers have been mostly focusing on toxicity assays. However, hepatic glucose production (HGP) and regulation have been largely neglected due to the lack of HGP assays in the field.

[0004] Current methods to evaluate and measure glucose production include, for example, the incorporation of radioactive and stable-labelled isotopes from substrates to glucose. The most widely used method is to measure the incorporation of deuterium from water into glucose. However, this method is more suitable for in vivo measurements and less so for in vitro measurements. This is because the quantification of the labelled isotopes is typically done using nuclear magnetic resonance (NMR) or mass spectrometry-based techniques, which are cumbersome, expensive, and less suitable for high-throughput screening assays. Therefore, there is a need for new methods and assays to monitor hepatic glucose production.SUMMARY

[0005] In one aspect, the present disclosure refers to a method of identifying a compound that affects hepatic glucose production, the method comprising: a) incubating a first and a second sample of hepatic cells in glucose production media, wherein the first sample comprises the compound and wherein the second sample does not comprise the compound; b) measuring glucose secreted by the hepatic cells in the first sample, thereby obtaining a first measurement; c) measuring glucose secreted by the hepatic cells in a second sample, thereby obtaining a secondmeasurement; and d) comparing the first measurement and the second measurement, wherein a difference in the first measurement and the second measurement indicates that the compound affects hepatic glucose production.

[0006] In another aspect, the present disclosure refers to a method of identifying a compound that increases or decreases hepatic glucose production due to gluconeogenesis, the method comprising: a) incubating a first, a second, a third, and a fourth sample of hepatic cells in glucose production media, wherein the first sample comprises the compound, wherein the second sample does not comprise the compound, wherein the first and second sample do not comprise a gluconeogenic substrate, wherein the third sample comprises the compound, wherein the fourth sample does not comprise the compound, and wherein the third and fourth sample comprise the gluconeogenic substrate; b) measuring glucose production by the first and the third samples of hepatic cells of step a) after incubation with the compound, comprising: i) measuring glucose secreted by the hepatic cells in the first sample, thereby obtaining a first measurement; ii) measuring glucose secreted by the hepatic cells in the third sample, thereby obtaining a third measurement; iii) subtracting the third measurement from the first measurement to obtain a first value of hepatic glucose production from gluconeogenesis after incubation of the hepatic cells with the compound; (c) measuring glucose production by the second and the fourth samples of hepatic cells of step a) after incubation without the compound, comprising: iv) measuring glucose secreted by the hepatic cells in the second sample, thereby obtaining a second measurement; v) measuring glucose secreted by the hepatic cells in the fourth sample, thereby obtaining a fourth measurement; vi) subtracting the fourth measurement from the second measurement to obtain a second value of hepatic glucose production from gluconeogenesis after incubation of the hepatic cells without the compound; and (d) comparing the first value obtained in step (b) with the second value obtained in step (c), wherein if the first value obtained in step (b) is greater than the second value obtained in step (c), the compound is identified to increase gluconeogenesis in hepatic cells, and wherein if the first value obtained in step (b) is less than the second value obtained in step (c), the compound is identified to decrease gluconeogenesis in hepatic cells.

[0007] In yet another aspect, the present disclosure refers to a method of identifying hepatic cells that have increased or decreased hepatic glucose production in response to treatment with a compound, the method comprising: (a) incubating a fifth and a sixth sample of hepatic cells in glucose production media, wherein the fifth sample comprises the compound and wherein the sixth sample does not comprise the compound; (b) measuring glucose secreted by the hepatic cell in the fifth sample, thereby obtaining a fifth measurement; (c) measuring glucose secreted by hepatic cells in the sixth sample, thereby obtaining a sixth measurement; (d) comparing the fifthmeasurement and the sixth measurement, wherein if the fifth measurement is greater than the sixth measurement, the hepatic cells are identified to have increased hepatic glucose production in response to treatment with the compound, and wherein if the fifth measurement is less than the sixth measurement, the hepatic cells are identified to have decreased hepatic glucose production in response to treatment with the compound.

[0008] In a further aspect, the present disclosure refers to a kit comprising a glucose production media, a buffer to wash cells, a cell lysis buffer, one or more solutions that couples proteins to a quantifiable tag, and instruction manual to measure hepatic glucose production from gluconeogenesis and / or glycogenolysis.

[0009] In another aspect, the present disclosure refers to a kit comprising a glucose production media, one or more stimulatory or inhibitory agents, a buffer to wash cells, a cell lysis buffer, one or more solutions that couples proteins to a quantifiable tag, and instruction manual to determine whether hepatic glucose production from gluconeogenesis and / or glycogenolysis is increased or decreased under stimulatory or inhibitory conditionsBRIEF DESCRIPTION OF THE DRAWINGS

[0010] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:

[0011] Figure 1 shows a schematic of the methodology of the hepatic glucose production (HGP) assay.

[0012] Figure 2 shows column graphs depicting measurements of hepatic glucose production (HGP) in hPSC-derived hepatocytes and mouse primary hepatocytes. Figure 2A shows results of a hepatic glucose production assay method performed with or without gluconeogenic substrates (pyruvate and lactate) on hPSC-derived hepatocytes. Figure 2B shows results of a hepatic glucose production assay method performed with or without gluconeogenic substrates (pyruvate and lactate) on mouse primary hepatocytes. Figure 2C shows the amount of gluconeogenesis in hPSC- derived hepatocytes. Figure 2D shows the amount of gluconeogenesis in mouse primary hepatocytes. The amount of glucose being produced in mouse primary hepatocytes is normalized to the total protein content. The amount of gluconeogenesis in Figures 2C and 2D was determined as the difference between the amount of glucose produced with or without gluconeogenic substrates. The hepatic glucose production assay was performed as described herein.

[0013] Figure 3 shows column graphs with results indicating that hepatocytes from Z / zxJ’7’ mice have lower hepatic glucose production (HGP). It is noted that ZHX3 is a gene that isassociated with fasting blood glucose levels, type 2 diabetes risk and hepatic glucose metabolism. Figures 3A and 3B show results of hepatic glucose production assays with or without gluconeogenic substrates (sodium pyruvate and sodium lactate) in hepatocytes isolated from wildtype (WT) andmice (n=4-5). Figure 3C shows images of periodic acid Schiff (PAS) staining performed on liver tissues obtained from WT and ZhxS’^ mice (n=3-5). Scale bar = 100 pm. Data presented as mean ± SEM. *, p<0.05. Statistical analyses were performed using unpaired t-tcst.

[0014] Figure 4 shows column graphs depicting the results of modulation of hepatic glucose production (HGP) by known molecules, such as, but not limited to, insulin (INS), and dexamethasone and forskolin (Dex+Fsk) in various liver models. Figure 4A shows results of performing the hepatic glucose production assay method on HepG2 cells. The amount of glucose being produced is normalized to the total protein content. Figure 4B shows results of performing the hepatic glucose production assay method on hPSC-derived hepatocytes. Figure 4C shows results of performing the hepatic glucose production assay on mouse primary hepatocytes.

[0015] Figure 5 shows column graphs depicting glucose concentrations per mg of protein obtained from performing the hepatic glucose production (HGP) assay method on HepG2 cells transfected with an empty vector control (i.e., a vector not comprising a gene of interest, used as a control), or transfected with a ZHX3 gene knockout vector, sgZHX3-2 or sgZHX3-9, each directed to different CR1SPR constructs that knockout the ZHX3 gene by targeting different sites. Under basal, non-treated conditions, the knockout of ZHX3 in the HepG2 cells increased HGP. Under treatment with insulin (INS), HGP was repressed in the empty vector control cells but not in the ZHX3 knockout cells, suggesting that ZHX3 could be mediating INS’s repression on HGP. Under treatment with dexamethasone (Dex) and forskolin (Fsk), HGP was increased in all cells.DEFINITIONS

[0016] As used herein, the term “hepatic cell” or “hepatic cells” includes, but is not limited to, hepatocytes, hepatoma cell lines, pluripotent stem cell-derived hepatocyte-like cells (PSC- hcpatocytcs), and pluripotent stem ccll-dcrivcd liver organoids. Hepatic cells as used herein refer to, but are not limited to, hepatic cells from mammals, including but not limited to, humans, mice, rats, hamsters, rabbits, guinea pigs, dogs, cats, monkeys, pigs, sheep, cows, horses. In one example, the hepatic cells disclosed herein can be, but are not limited to, human hepatocytes, human hepatoma cell lines, human pluripotent stem cell-derived hepatocyte-like cells (hPSC- hepatocytes), human pluripotent stem cell-derived liver organoids, or combinations thereof.

[0017] As used herein, the term “hepatic glucose production” or “HGP” refers to an amount or concentration of glucose secreted by hepatic cells, and can include, for example, total hepatic glucose production, glucose production due to glycogenolysis, and / or hepatic glucose production due to gluconeogenesis. The term “total hepatic glucose production” refers to an amount of glucose produced by hepatic cells due to both glycogenolysis and gluconeogenesis pathways.

[0018] As used herein, the term “hepatic glucose production assay” or “HGP assay” refers to in vitro methods or assays disclosed herein in which samples of hepatic cells arc tested for their capability to produce glucose. Such methods can include, but are not limited to, incubating cells in glucose production media under different conditions, with or without gluconeogenic substrates, and / or with or without a compound of interest. Methods can include steps of obtaining the results of hepatic glucose production under the different conditions and comparing the same. Basis of comparison can be, but is not limited to, untreated hepatic cells, non-hepatic cells, knockout hepatic cells, or modified hepatic cells. It is noted that the term “HGP assay” is used interchangeably herein with the terms “method’, “screening method”, and “HGP assay method”. Although the methods described herein are referred to as hepatic glucose production methods or assays for screening hepatic glucose production in hepatic cells, a person skilled in the art would appreciate that the same methods or assays can be used to screen for glucose production in non- hepatic cells. In one example, the methods and assays disclosed herein can be used to screen for glucose production in a glucose-producing cell.

[0019] As used herein, the term “change” or “difference” refers to a relative alteration of an amount of glucose production, including an increase or decrease, in a sample of cells in comparison to a reference amount of glucose production in a reference sample of cells. In one example, the term “change” or “difference” refers to a relative alteration of an amount of hepatic glucose production, including an increase or decrease, in a sample of hepatic cells in comparison to a reference amount of hepatic glucose production in a reference sample of hepatic cells or non- hepatic cells. In one example, the “change” or “difference” refers to a statistically significant change or difference. In another example, the “change” or “difference” refers to a statistically significant change or difference, with a p value of p<0.05. In one example, the “change” or “difference” refers to a change or difference by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20% compared to a reference amount of glucose production by a reference sample of cells. In one example, the “change” or “difference” refers to an change or difference by at least 0.1 pg, at least 0.2 pg, at least 0.3 pg, at least 0.4 pg, at least 0.5 pg, at least 0.6 pg, at least 0.7 pg, at least 0.8 pg, at least0.9 pg, at least 1.0 pg, at least 1.1 pg, at least 1.2 pg, at least 1.3 pg, at least 1.4 pg, at least 1.5 pg, at least 1.6 pg, at least 1.7 pg, at least 1.8 pg, at least 1.9 pg, at least 2.0 pg, at least 2.1 pg, at least 2.2 pg, at least 2.3 pg, at least 2.4 pg, or at least 2.5 pg of glucose production compared to a reference amount of glucose production by a reference sample of cells. In one example, the “change” or “difference” refers to an change or difference by at least 0.01 pg / mg, at least 0.02 pg / mg, at least 0.03 pg / mg, at least 0.04 pg / mg, at least 0.05 pg / mg, at least 0.06 pg / mg, at least 0.07 pg / mg, at least 0.08 pg / mg, at least 0.09 pg / mg, at least 0.1 pg / mg, at least 0.2 pg / mg, at least 0.3 pg / mg, at least 0.4 pg / mg, at least 0.5 pg / mg, at least 0.6 pg / mg, at least 0.7 pg / mg, at least 0.8 pg / mg, at least 0.9 pg / mg, at least 1.0 pg / mg, at least 1.5 pg, at least 2.0 pg, or at least 2.5 pg of glucose production per mg of protein compared to a reference amount of glucose production per mg of protein by a reference sample of cells.10020 J In another example a “change” or “difference” can also be provided using the term “fold change”, whereby it is defined as the ratio between the two quantities; for quantities X and Y, the fold change of Y with respect to X is Y / X. In other words, a change from 30 to 60 is defined as a fold-change of 2. This is also referred to as a “2-fold increase” Similarly, a change from 30 to 15 is referred to as a “2-fold decrease”.

[0021] As used herein, the terms “increase” and “decrease” refer to the relative alteration of an amount of glucose production of a sample of cells in comparison to a reference amount of glucose production of a reference sample of cells. In one example, the terms “increase” and “decrease” refer to the relative alteration of an amount of hepatic glucose production of a sample of hepatic cells in comparison to a reference amount of hepatic glucose production of a reference sample of hepatic cells or non-hepatic cells. An increase thus indicates a change on a positive scale, whereas a decrease indicates a change on a negative scale. In one example, the “increase” refers to an increase of at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20% compared to a reference amount of glucose production by a reference sample of cells. In one example, the “increase” refers to an increase of at least 0.1 pg, at least 0.2 pg, at least 0.3 pg, at least 0.4 pg, at least 0.5 pg, at least 0.6 pg, at least 0.7 pg, at least 0.8 pg, at least 0.9 pg, at least 1.0 pg, at least 1.1 pg, at least 1.2 pg, at least 1.3 pg, at least 1.4 pg, at least 1.5 pg, at least 1.6 pg, at least 1.7 pg, at least 1.8 pg, at least 1.9 pg, at least 2.0 pg, at least 2.1 pg, at least 2.2 pg, at least 2.3 pg, at least 2.4 pg, or at least 2.5 pg of glucose production compared to a reference amount of glucose production by a reference sample of cells. In one example, the “increase” refers to an increase of at least 0.01 pg / mg, at least 0.02 pg / mg, at least 0.03 pg / mg, at least 0.04 pg / mg, at least 0.05 pg / mg, at least 0.06 pg / mg, at least0.07 pg / mg, at least 0.08 pg / mg, at least 0.09 pg / mg, at least 0.1 pg / mg, at least 0.2 pg / mg, at least 0.3 pg / mg, at least 0.4 pg / mg, at least 0.5 pg / mg, at least 0.6 pg / mg, at least 0.7 pg / mg, at least 0.8 pg / mg, at least 0.9 pg / mg, at least 1.0 pg / mg, at least 1.5 pg, at least 2.0 pg, or at least 2.5 pg of glucose production per mg of protein compared to a reference amount of glucose production per mg of protein by a reference sample of cells. In one example, the “increase” refers to a statistically significant increase. In another example, the “increase” refers to a statistically significant increase, with a p value of p<0.05. In one example, the “decrease” refers to an decrease of at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20% compared to a reference amount of glucose production by a reference sample of cells. In one example, the “decrease” refers to an decrease of at least 0.1 pg, at least 0.2 pg, at least 0.3 pg, at least 0.4 pg, at least 0.5 pg, at least 0.6 pg, at least 0.7 pg, at least 0.8 pg, at least 0.9 pg, at least 1.0 pg, at least 1.1 pg, at least 1.2 pg, at least 1.3 pg, at least 1.4 pg, at least 1.5 pg, at least 1.6 pg, at least 1.7 pg, at least 1.8 pg, at least 1.9 pg, at least 2.0 pg, at least 2.1 pg, at least 2.2 pg, at least 2.3 pg, at least 2.4 pg, or at least 2.5 pg of glucose production compared to a reference amount of glucose production by a reference sample of cells. In one example, the “decrease” refers to an decrease of at least 0.01 pg / mg, at least 0.02 pg / mg, at least 0.03 pg / mg, at least 0.04 pg / mg, at least 0.05 pg / mg, at least 0.06 pg / mg, at least 0.07 pg / mg, at least 0.08 pg / mg, at least 0.09 pg / mg, at least 0.1 pg / mg, at least 0.2 pg / mg, at least 0.3 pg / mg, at least 0.4 pg / mg, at least 0.5 pg / mg, at least 0.6 pg / mg, at least 0.7 pg / mg, at least 0.8 pg / mg, at least 0.9 pg / mg, at least 1.0 pg / mg, at least 1.5 pg, at least 2.0 pg, or at least 2.5 pg of glucose production per mg of protein compared to a reference amount of glucose production per mg of protein by a reference sample of cells. In one example, the “decrease” refers to a statistically significant decrease. In another example, the “decrease” refers to a statistically significant decrease, with a p value of p<0.05.

[0022] As used herein, the term “stimulatory agent” is interchangeably used with the term “activator”. As used herein, the term “stimulatory agent” refers to a compound that stimulatcs / activatcs a signalling pathway or part thereof to increase hepatic glucose production. In one example, a stimulatory agent can activate the cyclic adenosine monophosphate (cAMP) signalling pathway. The stimulatory agent can increase hepatic glucose production due to glycogenolysis and / or gluconeogenesis.

[0023] As used herein, the term “inhibitory agent” is interchangeably used with the term “repressor”. As used herein, the term “inhibitory agent” refers to a compound that inhibits / represses a signalling pathway, or part thereof, to decrease hepatic glucose production. Inone example, the inhibitor}' agent can inhibit the glucagon signalling pathway. The inhibitory agent can decrease hepatic glucose production due to glycogenolysis and / or gluconeogenesis.DETAILED DESCRIPTION[0024 J The present disclosure refers to assay methods for evaluating hepatic glucose production (HGP). The assay methods can be used for evaluating HGP in hepatocytes, human pluripotent stem ccll-dcrivcd hcpatocytc-likc cells (hPSC-hcpatocytcs), and human pluripotent stem cell- derived liver organoids. The assay methods can be used to determine whether HGP is performed by glycogenolysis or gluconeogenesis. The assay methods can also be used to evaluate the modulation of HGP under stimulatory and inhibitory conditions, or modulation of HGP in response to a substance of interest. It can be appreciated that while the present disclosure refers to hepatic glucose production assays for screening hepatic glucose production in hepatic cells, the assays or methods disclosed herein can also be used to evaluate (non-hepatic) glucose production in non- hcpatic cells, particularly, glucose -producing non-hepatic cells.

[0025] Studying hepatic glucose production and differentiating between glucose produced from glycogen storage (due to glycogenolysis) and glucose produced from substrates such as pyruvate and lactate (due to gluconeogenesis) is important for understanding the mechanisms governing HGP, for identifying which HGP pathway is affected by a disease, and for discovering therapeutic agents and their effectiveness in regulating HGP or targeting a specific HGP pathway.

[0026] The methods disclosed herein (also interchangeably referred to as the “HGP assay” or “screening method”) evaluate the ability of hepatic cells to produce glucose from glycogen storage (due to glycogenolysis) and from gluconeogenic substrates (due to gluconeogenesis) in vitro. Additionally, the methods disclosed herein measures a change in total hepatic glucose production, a change in hepatic glucose production from glycogenolysis, and / or a change in hepatic glucose production from gluconeogenesis. In one example, the methods disclosed herein measures an increase or decrease in hepatic glucose production, including an increase or decrease in total hepatic glucose production, an increase or decrease in hepatic glucose production from glycogenolysis, and / or an increase or decrease in hepatic glucose production from gluconeogenesis.

[0027] In one example, the change in hepatic glucose production is compared to the hepatic glucose production of a reference sample. Thus, in one example, a reference control can be either a positive or negative control, as the case may be. A person skilled in the art would appreciate how to determine which is required. In one example, the reference sample is a sample of hepatic cells. In one example, the reference sample can be a wild-type, healthy, non-diseased and / or untreated,sample of hepatic cells. In one example, the reference sample is a sample of non-hepatic cells. The term “test sample” and “sample” can be used interchangeably in the context of the present disclosure. In one example, the reference sample can be a cell that does not produce glucose. In one example, the reference sample of hepatic cells can be a sample obtained at one time point. In another example, the reference sample of hepatic cells can be a sample obtained prior to treatment. In another example, the reference sample of hepatic cells can be hepatic cells incubated in glucose production media supplemented with a gluconeogenic substrate. In another example, the reference sample of hepatic cells can be hepatic cells incubated in glucose production media not supplemented with a gluconeogenic substrate. In another example, the reference sample of hepatic cells can be hepatic cells incubated in glucose production media supplemented with a compound. In another example, the reference sample of hepatic cells can be hepatic cells incubated in glucose production media not supplemented with a compound. In another example, the reference sample of hepatic cells can be hepatic cells incubated in glucose production media supplemented with a gluconeogenic substrate and a compound. In another example, the reference sample of hepatic cells can be hepatic cells incubated in glucose production media supplemented with a gluconeogenic substrate, without a compound. In another example, the reference sample of hepatic cells can be hepatic cells incubated in glucose production media supplemented with a compound, without a gluconeogenic substrate.[0028 J In another example, the methods disclosed herein measures an increase or decrease in hepatic glucose production in mutant or diseased hepatic cells compared to a reference amount of hepatic glucose production measured from a reference sample of hepatic cells.

[0029] The methods disclosed herein can be performed on various hepatic cells, including but not limited to, primary hepatocytes, hepatoma cell lines, pluripotent stem cell-derived hepatocytelike cells (PSC-hepatocytes) and pluripotent stem cell-derived liver organoids. In one example, the methods disclosed herein are performed on human primary hepatocytes, human hepatoma cell lines, human pluripotent stem cell-derived hepatocyte -like cells (hPSC-hepatocytes) and human pluripotent stem cell-derived liver organoids. In another example, the methods disclosed herein arc performed on hepatic cells from mice, rats, hamsters, rabbits, guinea pigs, dogs, cats, monkeys, pigs, sheep, cows, horses. In one example, the cells are hepatocytes. In one example, the cells are hepatoma cells. In one example, the cells are HepG2 cells. In one example, the cells are hPSC- derived hepatocytes. In one example, the cells are mouse hepatocytes. In one example, the cells are ZHX3 knockout HepG2 cells.

[0030] The hepatocyte cell line includes but is not limited to AML12 and THLE-2. The hepatoma cell line includes but is not limited to, HepG2, Hep3B, HepaRG, Huh7, HCC, and HB611.

[0031] In another example, the methods disclosed herein can be performed on non-hepatic cells. In another example, the methods disclosed herein can be performed on glucose-producing cells. In one example, the non-hepatic cells are renal cortex cells. In one example, the glucose- producing cells arc from mice, rats, hamsters, rabbits, guinea pigs, dogs, cats, monkeys, pigs, sheep, cows, horses.

[0032] In one example, the methods disclosed herein evaluate a change in hepatic glucose production under stimulatory (e.g., from glucocorticoid and glucagon signalling) conditions. Stimulatory conditions as described herein refer to incubation of a sample of hepatic cells in glucose production media supplemented with a stimulatory agent. In another example, the methods disclosed herein evaluate whether a sample of hepatic cells is responsive to a stimulatory agent.

[0033] In one example, the stimulatory agent is a compound that increases glucocorticoid signalling. In another example, the stimulatory agent is a compound that increases glucagon signalling. In yet another example, the stimulatory agent is a compound that increases the production of cyclic adenosine monophosphate AMP (cAMP). In a further example, the stimulatory agent is a compound selected from the group consisting of forskolin, dexamethasone, dibutyryl-cyclic AMP, and 3 -isobutyl- 1 -methylxanthine (LB MX). In one example, the compound is dexamethasone. In one example, the compound is forskolin. In one example, the compound is dexamethasone, forskolin, or a combination thereof.

[0034] In one example, the methods disclosed herein evaluate a change in hepatic glucose production under and inhibitory (e.g., from insulin signalling) conditions. Inhibitory conditions as described herein refer to incubation of a sample of hepatic cells in glucose production media supplemented with an inhibitory agent. In another example, the methods disclosed herein evaluate whether a sample of hepatic cells is responsive to an inhibitory agent.

[0035] In one example, the inhibitory agent is a compound that increases insulin signalling. In another example, the inhibitory agent is a hypoglycacmic compound. In yet another example, the inhibitory agent is a compound selected from the group consisting of insulin, metformin, glyburide, and pioglitazone. In one example, the compound is insulin.

[0036] In one example, the method disclosed herein is a method of identifying a compound that increases or decreases hepatic glucose production, the method comprising: (a) incubating a first and a second sample of hepatic cells in glucose production media, wherein the first sample comprises the compound and wherein the second sample does not comprise the compound; (b)measuring glucose secreted by the hepatic cells in the first sample, thereby obtaining a first measurement; (c) measuring glucose secreted by the hepatic cells in a second sample, thereby obtaining a second measurement; and (d) comparing the first measurement and the second measurement, wherein if the first measurement is greater than the second measurement, the compound is identified to increase hepatic glucose production, and wherein if the first measurement is less than the second measurement, the compound is identified to decrease hepatic glucose production.

[0037] In another example, the method disclosed herein is a method of identifying a compound that increases or decreases hepatic glucose production due to gluconeogenesis, the method comprising: (a) incubating a first, a second, a third, and a fourth sample of hepatic cells in glucose production media, wherein the first sample comprises the compound, wherein the second sample does not comprise the compound, wherein the first and second sample do not comprise a gluconeogenic substrate, wherein the third sample comprises the compound, wherein the fourth sample docs not comprise the compound, and wherein the third and fourth sample comprise the gluconeogenic substrate; (b) measuring glucose production by the first and the third samples of hepatic cells of step a) after incubation with the compound, comprising: i) measuring glucose secreted by the hepatic cells in the first sample, thereby obtaining a first measurement; ii) measuring glucose secreted by the hepatic cells in the third sample, thereby obtaining a third measurement; iii) subtracting the third measurement from the first measurement to obtain a first value of hepatic glucose production due to gluconeogenesis after incubation of the hepatic cells with the compound; (c) measuring glucose production by the second and the fourth samples of hepatic cells of step a) after incubation without the compound, comprising; iv) measuring glucose secreted by the hepatic cells in the second sample, thereby obtaining a second measurement; v) measuring glucose secreted by the hepatic cells in the fourth sample, thereby obtaining a fourth measurement; vi) subtracting the fourth measurement from the second measurement to obtain a second value of hepatic glucose production due to gluconeogenesis after incubation of the hepatic cells without the compound; and (d) comparing the first value obtained in step (b) with the second value obtained in step (c), wherein if the first value obtained in step (b) is greater than the second value obtained in step (c), the compound is identified to increase gluconeogenesis in hepatic cells, and wherein if the first value obtained in step (b) is less than the second value obtained in step (c), the compound is identified to decrease gluconeogenesis in hepatic cells.

[0038] In another example, the method disclosed herein is a method of identifying hepatic cells that have increased or decreased hepatic glucose production in response to treatment with a compound, the method comprising: (a) incubating a fifth and a sixth sample of hepatic cells inglucose production media, wherein the fifth sample comprises the compound and wherein the sixth sample does not comprise the compound; (b) measuring glucose secreted by the hepatic cells in the fifth sample, thereby obtaining a fifth measurement; (c) measuring glucose secreted by the hepatic cells in the sixth sample, thereby obtaining a sixth measurement; and (d) comparing the fifth measurement and the sixth measurement, wherein if the fifth measurement is greater than the sixth measurement, the hepatic cells axe identified to have increased hepatic glucose production in response to treatment with the compound, and wherein if the fifth measurement is less than the sixth measurement, the hepatic cells are identified to have decreased hepatic glucose production in response to treatment with the compound.

[0039] In another example, the method disclosed herein is a method of identifying hepatic cells that have increased or decreased hepatic glucose production due to gluconeogenesis in response to treatment with a compound, the method comprising: (a) incubating a first, a second, a third, and a fourth sample of hepatic cells in glucose production media, wherein the fifth sample comprises the compound, wherein the sixth sample docs not comprise the compound, wherein the fifth and sixth samples do not comprise a gluconeogenic substrate, wherein the seventh sample comprises the compound, wherein the eighth sample does not comprise the compound, and wherein the seventh and eighth samples comprise a gluconeogenic substrate; (b) measuring glucose production by hepatic cells after incubation with the compound, comprising: i) measuring glucose secreted by the hepatic cell in the fifth sample, thereby obtaining a fifth measurement; ii) measuring glucose secreted by the hepatic cells in the seventh sample, thereby obtaining a seventh measurement; iii) subtracting the seventh measurement from the fifth measurement to obtain a first value of hepatic glucose production due to gluconeogenesis after incubation of the hepatic cells with the compound; (c) measuring glucose production by hepatic cells after incubation without the compound, comprising: iv) measuring glucose secreted by the hepatic cells in the sixth sample, thereby obtaining a sixth measurement; v) measuring glucose secreted by the hepatic cells in the eighth sample, thereby obtaining a eighth measurement; vi) subtracting the eighth measurement from the sixth measurement to obtain a second value of the hepatic glucose production due to gluconeogenesis after incubation of the hepatic cells without the compound; and (d) comparing the first value obtained in step (b) with the second value obtained in step (c), wherein if first value obtained in step (b) is greater than the second value obtained in step (c), the hepatic cells are identified to have increased gluconeogenesis in response to treatment with the compound, and wherein if the first value obtained in step (b)is less than the second value obtained in step (c), the hepatic cells are identified to have decreased gluconeogenesis in response to treatment with the compound.

[0040] In one example, the compound as used in the methods disclosed herein is a stimulatory agent or an inhibitory agent.

[0041] In another example, the compound as used in the methods disclosed herein is a stimulatory agent. In yet another example, the stimulatory agent is selected from the group consisting of forskolin, dexamethasone, dibutyryl-cyclic AMP, and 3-isobutyl-l- methylxanthine (IB MX).

[0042] In another example, the compound as used in the methods disclosed herein is an inhibitory agent. In yet another example, the inhibitory agent is a hypoglycaemic compound. In yet another example, the inhibitory agent is selected from the group consisting of insulin, metformin, glyburide and pioglitazone.

[0043] In another example, the hepatic cells as used in the methods disclosed herein are selected from the group consisting of primary hepatocytes, liver organoids, HepG2 cells, human hepatic cancer cells, human pluripotent stem cell (hPSC) -derived hepatocyte-like cells, human pluripotent stem cell (hPSC)-dcrivcd hepatocytes, and human pluripotent stem cell (hPSC)-dcrivcd liver organoids. In yet another example, the hepatic cells as used in the methods disclosed herein are genetically modified.

[0044] In another example, the methods as disclosed herein test one or more compounds. In yet another example, the one or more compounds as used in the methods disclosed herein are tested simultaneously.

[0045] In one example, the methods as disclosed herein comprises an incubating step. In another example, the incubating step in the methods disclosed herein is performed for a period of time between 5 to 8 hours. In another example, the incubating step in the methods disclosed herein is performed for a period of time of 6 hours. In yet another example, the incubating step in the methods disclosed herein is between 5 to 8 hours, or 6 hours.

[0046] In another example, the methods as disclosed herein comprises a step of normalizing the amount of glucose measured in a sample to the total protein content of the same sample.

[0047] In one example, the samples of hepatic cells used in the methods disclosed herein are scrum-starvcd for a period of time prior to the incubation step. In another example, the samples of hepatic cells used in the methods disclosed herein are serum-starved overnight prior to the incubation step. In another example, the samples of hepatic cells used in the methods disclosed herein are serum-starved overnight at 37 °C. In another example, the samples of hepatic cells used in the methods disclosed herein are serum-starved overnight at 37 °C and 5 % CO2. In yet another example, the samples of hepatic cells used in the methods disclosed herein are washed after being serum-starved and prior to the incubation step. In yet another example, the samples of hepatic cellsused in the methods disclosed herein are washed with a buffer to remove residual glucose after being serum-starved and prior to the incubation step.

[0048] In one example, the glucose production media used in the methods disclosed herein do not comprise glucose. In one example, the glucose production media used in the methods disclosed herein do not comprise serum. In one example, the glucose production media used in the methods disclosed herein do not comprise phenol red. In one example, the glucose production media used in the methods disclosed herein do not comprise pyruvate. In one example, the glucose production media used in the methods disclosed herein do not comprise glucose, serum, phenol red, pyruvate, or any combination thereof. In one example, the glucose production media used in the methods disclosed herein do not comprise glucose, serum, phenol red, and pyruvate.

[0049] In another example, the gluconeogenic substrate used in the methods disclosed herein is pyruvate. In another example, the gluconeogenic substrate used in the methods disclosed herein is lactate. In another example, the gluconeogenic substrate used in the methods disclosed herein is sodium pyruvate. In another example, the gluconeogenic substrate used in the methods disclosed herein is sodium lactate. In another example, the gluconeogenic substrate used in the methods disclosed herein is pyruvate and lactate. In one example, the gluconeogenic substrate used in the methods disclosed herein is sodium pyruvate, sodium lactate, or a combination thereof. In another example, the gluconeogenic substrate used in the methods disclosed herein is sodium pyruvate and sodium lactate. In another example, the gluconeogenic substrate used in the methods disclosed herein is glycerol. In another example, the gluconeogenic substrate used in the methods disclosed herein is one or more gluconeogenic amino acid.

[0050] In one example, the amount of glucose measured in the methods disclosed herein is measured fluorometrically, photometrically, colorimetrically, or spectrophotometrically.

[0051] In one example, the method disclosed herein is a method of identifying hepatic cells of a test sample that have increased or decreased hepatic glucose production compared to a reference sample of hepatic cells, the method comprising: (a) incubating a test and a reference sample of hepatic cells in glucose production media; (b) measuring glucose secreted by the hepatic cells in the test sample, thereby obtaining a test measurement; (c) measuring glucose secreted by the hepatic cells in the reference sample, thereby obtaining a reference measurement; and (d) comparing the test measurement and the reference measurement, wherein if the test measurement is greater than the reference measurement, the hepatic cells of the test sample are identified to have increased hepatic glucose production compared to the reference sample of hepatic cells, and wherein if the test measurement is less than the reference measurement, the hepatic cells of thetest sample are identified to have decreased hepatic glucose production compared to the reference sample of hepatic cells.

[0052] In another example, the method disclosed herein is a method of identifying hepatic cells of a test sample that have increased or decreased hepatic glucose production from gluconeogenesis compared to hepatic cells of a reference sample, the method comprising: (a) incubating a first test sample and a second test sample of hepatic cells in glucose production media, wherein the first test sample comprises a gluconeogenic substrate, wherein the second test sample docs not comprise the gluconeogenic substrate, and incubating a first reference sample and a second reference sample of hepatic cells, wherein the first reference sample comprises the gluconeogenic substrate, and wherein the second reference sample does not comprise the gluconeogenic substrate; (b) measuring glucose production by hepatic cells from the test sample, comprising: i) measuring glucose secreted by the hepatic cells in the first test sample, thereby obtaining a first test measurement; ii) measuring glucose secreted by the hepatic cells in the second test sample, thereby obtaining a second test measurement; iii) subtracting the second test measurement from the first test measurement to obtain a test value of hepatic glucose production from gluconeogenesis of the test sample of hepatic cells; (c) measuring glucose production by hepatic cells from the reference sample, comprising: iv) measuring glucose secreted by the hepatic cells in the first reference sample, thereby obtaining a first reference measurement; v) measuring glucose secreted by the hepatic cells in the second reference sample, thereby obtaining a second reference measurement; vi) subtracting the second reference measurement from the first reference measurement to obtain a reference value of hepatic glucose production from gluconeogenesis of the reference sample of hepatic cells; and (d) comparing the test value obtained in step (b) with the reference value obtained in step (c), wherein if the test value obtained in step (b) is greater than the reference value obtained in step (c), the test sample is identified to have increased gluconeogenesis compared to the reference sample, and wherein if the test value obtained in step (b) is less than the reference value obtained in step (c), the test sample is identified to have decreased gluconeogenesis compared to the reference sample.

[0053] In one example, the test sample as used in the method disclosed herein is selected from the group consisting of wild-type hepatic cells, hepatic cells obtained from a diseased individual, hepatic cells obtained from an individual having diabetes, hepatic cells transfected with a viral vector, hepatic cells transfected with a vector that knocks out an endogenous gene, and genetically modified hepatic cells.

[0054] In one example, the reference sample as used in the method disclosed herein is selected from the group consisting of: wild-type hepatic cells, hepatic cells obtained from a healthyindividual, and hepatic cells obtained from a non-diseased individual. In one example, the reference sample as used in the method disclosed herein is a non-hepatic cell. In another example, the reference sample as used in the method disclosed herein is a cell that does not produce glucose.

[0055] In one example, the reference sample and test sample are obtained from the same individual, wherein the reference sample is a sample obtained at one time point, and the test sample is obtained at a second time point. In another example, the reference sample and test sample axe obtained from the same individual, wherein the reference sample is a sample obtained prior to treatment, and wherein the test sample is a sample obtained after treatment.

[0056] In another example, the disclosure refers to a kit comprising a glucose production media, a buffer to wash cells, a cell lysis buffer, one or more solutions that couples proteins to a quantifiable tag, and instruction manual to measure hepatic glucose production due to gluconeogenesis and / or glycogenolysis.

[0057] In another example, the disclosure refers to a kit comprising a glucose production media, one or more stimulatory or inhibitory agents, a buffer to wash cells, a cell lysis buffer, one or more solutions that couples proteins to a quantifiable tag, and instruction manual to determine whether hepatic glucose production from gluconeogenesis and / or glycogenolysis is increased or decreased under stimulatory or inhibitory conditions.

[0058] In one example, the kit disclosed herein is for use in a method as disclosed herein. In another example, the kit disclosed herein is for use in a method of identifying a compound that increases or decreases hepatic glucose production. In another example, the kit disclosed herein is for use in a method of identifying hepatic cells that have increased or decreased hepatic glucose production in response to treatment with a stimulatory agent or an inhibitory agent.Hepatic Glucose Production (HGP) Assay

[0059] The present disclosure refers to a HGP assay method, which is interchangeably referred to herein as a “method of screening” or “assay method” or “method”. The method disclosed herein can be performed in a standard laboratory setting. In the method disclosed herein, the hepatic cells are serum- starved. The hepatic cells can be serum-starved in Dulbecco’s Modified Eagle Medium (DMEM). In one example, the hepatic cells can be scrum-starved for a period of time between 1 to 24 hours, 2 to 24 hours, 3 to 24 hours, 4 to 24 hours, 5 to 24 hours, 6 to 24 hours, 7 to 24 hours, 8 to 24 hours, 9 to 24 hours, 10 to 24 hours, 11 to 24 hours, 12 to 24 hours, 13 to 24 hours, 14 to 24 hours, 15 to 24 hours, 16 to 24 hours, 17 to 24 hours, 18 to 24 hours, 19 to 24 hours, 20 to 24 hours, 21 to 24 hours, 22 to 24 hours, or 23 to 24 hours. In one example, the hepatic cells can be serum-starved for a period of time between 1 to 23 hours, 1 to 22 hours, 1 to 21 hours, 1 to 20 hours, 1 to 19 hours, 1 to 18 hours, 1 to 17 hours, 1 to 16 hours, 1 to 15 hours, 1 to 14 hours, 1 to13 hours, 1 to 12 hours, 1 to 11 hours, 1 to 10 hours, 1 to 9 hours, 1 to 8 hours, 1 to 7 hours, 1 to 6 hours, 1 to 5 hours, 1 to 4 hours, 1 to 3 hours, or 1 to 2 hours. In another example the hepatic cells can be serum-starved overnight. In another example, the hepatic cells can be serum-starved for about 24 hours, about 23 hours, about 22 hours, about 21 hours, about 20 hours, about 19 hours, about 18 hours, about 17 hours, about 16 hours, about 15 hours, about 14 hours, about 13 hours, about 12 hours, about 11 hours, about 10 hours, about 9 hours, about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, or about 1 hour.

[0060] In one example, the hepatic cells can be serum-starved at a temperature of between 30 to 40°C, between 31 to 40°C, between 32 to 40°C, between 33 to 40°C, between 34 to 40°C, between 35 to 40°C, between 36 to 40°C, between 37 to 40°C, between 38 to 40°C, or between 39 to 40°C. In one example, the hepatic cells can be serum-starved at a temperature of between 30 to 39°C, between 30 to 38°C, between 30 to 37°C, between 30 to 36°C, between 30 to 35°C, between 30 to 34°C, between 30 to 33°C, between 30 to 32°C, or between 30 to 31°C. In another example, the hepatic cells can be scrum-starved at a temperature of about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, or about 40°C. In another example, the hepatic cells can be serum-starved at a temperature of about 37.0°C.

[0061] In one example, the hepatic cells are serum-starved at a CO2 percentage of between 3 to 8%, between 4 to 8%, between 5 to 8%, between 6 to 8%, or between 7 to 8%. In one example, the hepatic cells are serum-starved at a CO2 percentage of between 3 to 7%, between 3 to 6%, between 3 to 5%, or between 3 to 4%. In one example, the hepatic cells are serum-starved at a CO2 percentage of about 3%, about 4%, about 5%, about 6 %, about 7%, or about 8%. In one example, the hepatic cells are serum-starved at a CO2 percentage of about 5.0%.

[0062] Following serum starvation, the hepatic cells are washed to remove residual glucose and subsequently incubated in glucose production media. In one example, the hepatic cells are washed once, twice, or three times. In another example, the hepatic cells are washed in phosphate buffered saline (PBS) once, twice, or three times. In one example, the hepatic cells are washed in PBS once.

[0063] In one example, the glucose production media is serum-free. In another example, the glucose production media is phenol red-free. In another example, the glucose production media is glucose-free, hi yet another example, the glucose production media is serum-free, phenol red-free, and glucose-free.

[0064] The incubation in glucose production media can be for a period of time between 1 to 10 hours, between 1 to 9 hours, between 1 to 8 hours, between 1 to 7 hours, between 1 to 6 hours, between 1 to 5 hours, between 1 to 4 hours, between 1 to 3 hours, or between 1 to 2 hours. In one example, the incubation in glucose production media can be for a period of time between 2 to 10hours, between 2 to 9 hours, between 2 to 8 hours, between 2 to 7 hours, between 2 to 6 hours, between 2 to 5 hours, between 2 to 4 hours, or between 2 to 3 hours. In one example, the incubation in glucose production media can be for a period of time between 3 to 10 hours, between 3 to 9 hours, between 3 to 8 hours, between 3 to 7 hours, between 3 to 6 hours, between 3 to 5 hours, between 3 to 4 hours. In one example, the incubation in glucose production media can be for a period of time between 4 to 10 hours, between 4 to 9 hours, between 4 to 8 hours, between 4 to 7 hours, between 4 to 6 hours, or between 4 to 5 hours. In one example, the incubation in glucose production media can be for a period of time between 5 to 10 hours, between 5 to 9 hours, between 5 to 8 hours, between 5 to 7 hours, or between 5 to 6 hours. In one example, the incubation in glucose production media can be for a period of time between 6 to 10 hours, between 6 to 9 hours, between 6 to 8 hours, or between 6 to 7 hours. In one example, the incubation in glucose production media can be for a period of time between 7 to 10 hours, between 7 to 9 hours, or between 7 to 8 hours. In one example, the incubation in glucose production media can be for a period of time between 8 to 10 hours, or between 8 to 9 hours. In one example, the incubation in glucose production media can be for a period of time between 9 to 10 hours.

[0065] In one example, the incubation in glucose production media can be for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, or about 10 hours. In one example, the incubation in glucose production media is for 6 hours.

[0066] In one example, the hepatic cells can be incubated at a temperature of between 30 to 40°C, between 31 to 40°C, between 32 to 40°C, between 33 to 40°C, between 34 to 40°C, between 35 to 40°C, between 36 to 40°C, between 37 to 40°C, between 38 to 40°C, or between 39 to 40°C. In one example, the hepatic cells can be incubated at a temperature of between 30 to 39 °C, between 30 to 38°C, between 30 to 37°C, between 30 to 36°C, between 30 to 35°C, between 30to 34°C, between 30 to 33°C, between 30 to 32°C, or between 30 to 31°C. In another example, the hepatic cells can be incubated at a temperature of about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, or about 40°C. In another example, the hepatic cells can be incubated at a temperature of about 37.0°C.

[0067] In one example, the hepatic cells are incubated at a CO2 percentage of between 3 to 8%, between 4 to 8%, between 5 to 8%, between 6 to 8%, or between 7 to 8%. In one example, the hepatic cells are incubated at a CO2 percentage of between 3 to 7%, between 3 to 6%, between 3 to 5%, or between 3 to 4%. In one example, the hepatic cells are incubated at a CO2 percentage of about 3%, about 4%, about 5%, about 6%, about 7%, or about 8%. In one example, the hepatic cells are incubated at a CO2 percentage of about 5.0%.

[0068] The glucose production media, in which the hepatic cells are incubated, can be free of a gluconeogenic substrate. In other words, the glucose production media does not comprise a gluconeogenic substrate. The glucose production media in which the hepatic cells are incubated, can be supplemented with a gluconeogenic substrate. In other words, the glucose production media comprises a gluconeogenic substrate. In one example, the gluconeogenic substrate is pyruvate or lactate, or a combination thereof. In another example, the gluconeogenic substrate is sodium pyruvate, or sodium lactate, or a combination thereof.

[0069] The glucose production media, in which the hepatic cells are incubated, can be free of any additional compounds. In other words, the glucose production media does not comprise a compound. The glucose production media, in which the hepatic cells are incubated, can be supplemented with one or more additional compounds. In other words, the glucose production media comprises one or more compounds. The compound can be a stimulatory agent or an inhibitory agent.

[0070] To measure the amount of glucose secreted by the hepatic cells, the media, in which the hepatic cells are incubated, is harvested and the amount of glucose in the harvested media is then measured. In one example, the media is diluted. In another example, the media is not diluted. The glucose concentration can be measured using any means known in the art, including but not limited to, fluorometric assays, photometric assays, colorimetric assays, and spectrophotometric assays.

[0071] In one example, the glucose concentration is measured by a fluorometric assay. The fluorometric assay utilises a reaction involving glucose, wherein the reaction is coupled to a fluorescence detection system. In one example, the fluorometric assays may couple glucose oxidation and hydrogen peroxide production with a fluorescent hydrogen peroxide detection system. The fluorescence assay can be a Glucose Assay Kit (Abeam) or Amplex Red Glucose / Glucose Oxidase Assay Kit (A22189; Invitrogen).

[0072] In one example, the glucose concentration is measured by a photometric assay. The photometric assay utilises a reaction involving glucose, wherein the reaction is coupled to a photometric, luminescence, or bioluminescence detection system. In one example, the photometric assay utilises a reaction involving glucose, wherein the reaction is coupled to a biolumincsccncc detection system. In one example, the bioluminescent assay can be one that couples glucose oxidation and NADH production with a bioluminescent NADH detection system. Glucose dehydrogenase uses glucose and NAD+ to produce NADH. In the presence of NADH, a proluciferin Reductase Substrate is converted by Reductase to luciferin, and the luciferin is oxidized in the presence of a luciferase enzyme to produce light, which can then be measured. The photometric assay can be a Glucose-Glo Assay (Promega).

[0073] In one example, the glucose concentration is measured by a colorimetric assay. The colorimetric assay utilises a reaction involving glucose, wherein the reaction is coupled to a colorimetric detection system. The colorimetric assay can be a Glucose Assay Kit (Abeam).

[0074] In one example, the glucose concentration is measured by a spectrophotometric assay. The spectrophotometric assay utilises a reaction involving glucose, wherein the reaction is coupled to a spectrophotometric detection system. In one example, the spectrophotometric assays may couple glucose oxidation and hydrogen peroxide production with a spectrophotometric hydrogen peroxide detection system. The spectrophotometric assay can be an Amplex Red Glucose / Glucose Oxidase Assay Kit (A22189; Invitrogen).

[0075] As the number of hepatic cells may differ from sample to sample, the amount of glucose measured from each sample would also vary and thus be incomparable. In order for the amount of glucose of each well to be appropriate for comparison, the amount of glucose is normalised to the total protein content of the hepatic cells in their respective samples.

[0076] To measure the protein content of the hepatic cells, the protein content is first isolated from the hepatic cells. To isolate the hepatic cells, the hepatic cells are separated from the media by centrifugation. The media supernatant is used for measuring hepatic glucose production as described herein. The remaining pelleted hepatic cells are washed once, twice, or three times. In another example, the hepatic cells are washed in phosphate buffered saline (PBS) once, twice, or three times.

[0077] After washing, the hepatic cells are lysed to release the protein contents of the cells; and subsequently, the protein content is isolated and subjected to a protein quantification assay. The protein content can be measured using any means known in the art, including but not limited to, bicinchoninic (BCA) Assay.Differentiating hepatic glucose production (HGP) from gluconeogenesis and glycogenolysis

[0078] The methods disclosed herein can measure the amount of glucose being produced from glycogenolysis and gluconeogenesis. Gluconeogenesis can be measured by calculating the difference between the amount of glucose secreted by hepatic cells after incubation in glucose production media supplemented with a gluconeogenic substrate and the amount of glucose secreted by hepatic cells after incubation in glucose production media not supplemented with a gluconeogenic substrate. Particularly, the amount of glucose secreted by hepatic cells after incubation in glucose production media supplemented with a gluconeogenic substrate is the total amount of glucose produced from both glycogenolysis and gluconeogenesis. The amount of glucose secreted by hepatic cells after incubation in glucose production media not supplemented with a gluconeogenic substrate is the amount of glucose produced from glycogenolysis. Therefore,the amount of glucose produced from gluconeogenesis can be calculated by subtracting the amount of glucose produced from glycogenolysis from the total amount of glucose.

[0079] In one example, the method disclosed herein had been performed on human pluripotent stem cell (hPSC)-derived hepatocytes and mouse primary hepatocytes (Figure 2). The first bar in each graph indicates the amount of glucose secreted by the hepatic cells after incubation in glucose production media not supplemented with gluconeogenic substrates, thus representing an amount of hepatic glucose production from glycogenolysis (Figures 2A and 2B). Incubation of the hepatic cells in the presence of gluconeogenic substrates, resulted in the amount of glucose secreted by both models being higher compared to incubation in the absence of gluconeogenic substrates (the second bar in each graph of Figures 2A and 2B), suggesting successful induction of gluconeogenesis. By calculating the difference in the amount of glucose secreted by hepatic cells after incubation in glucose production media supplemented with or not supplemented with gluconeogenic substrates, the amount of hepatic glucose production due to gluconeogenesis can be determined (Figures 2C and 2D). The same pipeline can be applied with the presence of a compound to determine if the compound gluconeogenesis.Hepatocytes from Zhx3' / ' mice have lower hepatic glucose production (HGP)

[0080] The HGP assay method disclosed herein can be used to identify whether an increase or decrease in hepatic glucose production is due to an increase or decrease in gluconeogenesis and / or due to an increase or decrease in glycogenolysis.

[0081] In one example, to ascertain that the reduced HGP in Zhx3 ' mice was due to reduced gluconeogenesis, hepatocytes were isolated from wild-type (WT) and ZhxS'^ mice and the HGP assay was performed. In the absence of gluconeogenic substrates (pyruvate and lactate), HGP is contributed solely by glycogenolysis while HGP consists of glycogenolysis and gluconeogenesis in the presence of gluconeogenic substrates. In the absence or presence of gluconeogenic substrates, hepatocytes from ZhxS^' mice produced less glucose than those from WT mice (Figures 3A and 3B), demonstrating reduced hepatic glycogenolysis and gluconeogenesis in Zhx3'f' mice. The amount of glycogen in hepatocytes from WT and Zhx3' / ' mice appeared to be similar as shown by periodic acid Schiff (PAS) staining (Figure 3C), suggesting that reduced hepatic glycogenolysis in Z v4mice was likely not due to a difference in the amount of glycogen storage but the rate of breakdown.Modulation of hepatic glucose production (HGP) by known compounds such as insulin (INS) and dexamethasone (Dex) + forskolin (Fsk)

[0082] The hepatic glucose production (HGP) assay method disclosed herein can be used to identify hepatic cell lines that are responsive to known compounds. In one example, the HGP assaymethod disclosed herein had been performed on the human hepatoma line HepG2, human pluripotent stem cell (hPSC)-derived hepatocyte-like cells, and mouse primary hepatocytes. Insulin (INS) is a known repressor of HGP, while dexamethasone (Dex) and forskolin (Fsk) are known activators of the glucagon signalling pathway, which is known to upregulate HGP. Hence, these compounds were used to exemplify that the HGP assay method described herein can be applied to various liver models to identify cell lines that are responsive to the compounds, but also can be applied to identify regulators of HGP.

[0083] The amount of glucose secreted by hepatic cells after incubation in glucose production media not supplemented with any additional compounds was measured (non-treated in Figures 4A, 4B and 4C). The amount of glucose secreted by hepatic cells after incubation in glucose production media supplemented with INS or Dex+Fsk treatment was measured. Subsequently, the difference between the amount of glucose before and after treatment with the compound(s) was measured to determine how much the compound(s) affect HGP.

[0084] In HcpG2 and hPSC-dcrivcd hepatocytes, the treatment with 100 nM INS decreased the amount of glucose secreted, 'hile 1 uM of Dex and 10 uM of Fsk increased the amount of glucose secreted (Figures 4A and 4B). Similarly, in mouse primary hepatocytes, 1 uM of Dex and 10 uM of Fsk increased HGP (Figure 4C).

[0085] Figures 4 and 5 show that INS, and Dex+Fsk can reproducibly elicit the expected response of HGP across numerous experiments and in different liver models, showing that this HGP assay method can work robustly and consistently.Modulation of hepatic glucose production (HGP) in genetically modified cell lines by known compounds such as insulin (INS) and dexamethasone (Dex) + forskolin (Fsk)

[0086] The hepatic glucose production (HGP) assay method disclosed herein can be used to identify genes that have a role in regulating HGP. In one example, the HGP assay method disclosed herein had been performed on the human hepatoma line HepG2. In one example, the HGP assay method disclosed herein had been performed on HepG2 cells transfected with an empty vector (empty vector control), and HepG2 cells each transfected with a CRISPR construct that knocked out the ZHX3 gene by targeting different sites (sgZHX3-2 and sgZHX3-9). Insulin (INS) is a known repressor of HGP, while dexamethasone (Dex) and forskolin (Fsk) are known activators of the glucagon signalling pathway, which is known to upregulate HGP. Hence, these compounds were used to exemplify that the HGP assay method described herein can be applied to hepatic cell lines in which a particular gene is modified, to determine if the modified gene is a regulator of HGP.

[0087] The amount of glucose in the HepG2 cells transfected with the ZHX3 knockout vectors, sgZHX3-2 and sgZHX3-9 was greater than in the HepG2 cells transfected with the empty vector.under basal, non-treated conditions (Figure 4). In HepG2 cells transfected with the empty vector, treatment with 100 nM INS decreased the amount of glucose secreted. However, in HepG2 cells transfected with sgZHX3-2 and -,gZI 1X3-9. treatment with 100 nM INS did not change the amount of glucose secreted (Figure 4). This suggests that ZHX3 could be mediating insulin’s repression on HGP, and thus suggests that ZHX3 may have a role in regulating HGP. When treated with 1 uM of Dex and 10 uM of Fsk, the HepG2 cells transfected with the empty vector and the HepG2 cells transfected with gZHX3-2 or sgZHX3-9 had increased HGP.Scalability of the hepatic glucose production (HGP) assay

[0088] It will be appreciated that the methods disclosed herein refer to HGP assays performed on cells cultured on assay plates. The HGP assays methods described herein refer to small-scale or large-scale HGP assays including but not limited to HGP assays performed on 6-well plates, 12-well plates, 24-well plates, 48-well plates, 96-well plates, 384-well plates, or 1536-well plates. It will also be appreciated that HGP assays performed on the different sized assay plates will be adapted as appropriate, with the number of cells and volume of media reduced proportionally, to increase the throughput of this assay for screening.

[0089] In one example, the HGP assay method disclosed herein is performed on a 6-well plate. As the media required for the HGP assay is < 5 u L. it will be appreciated that the HGP assay can be performed in a well of a 96-well plate. The collection of media after 6 hours of incubation and performing of the HGP assay can all be done in a 96-well plate, allowing the use of multichannel pipettes for higher throughput. After the collection of media for glucose measurement, the remaining cells in the 96-well plate can be lysed directly in the plate, allowing high-throughput protein quantification using the bicinchoninic acid (BCA) assay for measuring the amount of protein.EXPERIMENTAL SECTIONHepatic glucose production assay

[0090] Hepatic cells were provided in 4 confluent wells in a 6-well plate. The hepatic cells were scrum-starved overnight at 37°C and 5% CO2. After scrum- starvation, the hepatic cells were washed with phosphate buffered saline (PBS) once to remove residual glucose. Subsequently, the hepatic cells were incubated in 1 ml of glucose production media for 6 hours at 37°C and 5% CO2. The glucose production media comprises 4 mM L-glutamine, and glucose-free, phenol red-free Dulbecco’s Modified Eagle Medium (DMEM), supplemented or not supplemented with gluconeogenic substrates (2 mM sodium pyruvate and 20 mM sodium lactate) (Figure 1).

[0091] Three of the four wells were supplemented with the following: gluconeogenic substrates (2 mM sodium pyruvate and 20 mM sodium lactate); gluconeogenic substrates and 1 pM dexamethasone and 10 pM forskolin (Dex+Fsk); and gluconeogenic substrates and 100 nM insulin (INS) (Figure 1).

[0092] After incubation in the glucose production media, the media was harvested to measure the amount of glucose secreted in each well. To harvest the media, the media from each well was centrifuged at 2000 rpm for 5 minutes to pellet any floating hepatic cells. The supernatant was then transferred into a new Eppendorf tube to be analysed. The amount of glucose in the supernatant was measured using Amplex Red Glucose / Glucose Oxidase Assay Kit (A22189) from Invitrogen.

[0093] For mouse hepatocytes, the media was diluted 50x for the analysis. For human hepatocytes, no dilution was performed.Normalizing amount of glucose to total protein content

[0094] Following measurement of glucose in the supernatant, the total protein content of the hepatic cells was measured. The pelleted hepatic cells were resuspended and washed once with phosphate buffered saline (PBS). Subsequently, the hepatic cells were lysed using 500 pL of mammalian protein extraction reagent (M-PER™). The lysate / cell suspension was then transferred into a new Eppendorf tube and vortexed vigorously before incubation on ice for 30 minutes. After incubation on ice, the lysate / cell suspension was centrifuged at 15000 rpm for 10 minutes at 4°C to pellet the cell debris. The supernatant was transferred into a new Eppendorf tube to be analysed. The amount of protein in the supernatant was measured using a BCA assay.

[0095] The invention illustratively described herein can suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications arc possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed can be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0096] As used in this application, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a genetic marker” includes a plurality of genetic markers, including mixtures and combinations thereof.

[0097] As used herein, the term “about”, in the context of concentrations of components of the formulations, typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically, + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0098] Throughout this disclosure, certain embodiments can be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0099] Certain embodiments can also be described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description of the embodiments with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0100] The invention has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0101] Other embodiments are within the following claims and non-limiting examples. In addition, where features or aspects of the invention arc described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Claims

CLAIMSWhat is claimed is:

1. A method of identifying a compound that affects hepatic glucose production, the method comprising: a) incubating a first and a second sample of hepatic cells in glucose production media, wherein the first sample comprises the compound and wherein the second sample does not comprise the compound; b) measuring glucose secreted by the hepatic cells in the first sample, thereby obtaining a first measurement; c) measuring glucose secreted by the hepatic cells in a second sample, thereby obtaining a second measurement; and d) comparing the first measurement and the second measurement, wherein a difference in the first measurement and the second measurement indicates that the compound affects hepatic glucose production.

2. A method of identifying a compound that increases or decreases hepatic glucose production due to gluconeogenesis, the method comprising: a) incubating a first, a second, a third, and a fourth sample of hepatic cells in glucose production media, wherein the first sample comprises the compound, wherein the second sample does not comprise the compound, wherein the first and second sample do not comprise a gluconeogenic substrate, wherein the third sample comprises the compound, wherein the fourth sample does not comprise the compound, and wherein the third and fourth sample comprise the gluconeogenic substrate; b) measuring glucose production by the first and the third samples of hepatic cells of step a) after incubation with the compound, comprising: i) measuring glucose secreted by the hepatic cells in the first sample, thereby obtaining a first measurement; ii) measuring glucose secreted by the hepatic cells in the third sample, thereby obtaining a third measurement; iii) subtracting the third measurement from the first measurement to obtain a first value of hepatic glucose production from gluconeogenesis after incubation of the hepatic cells with the compound;(c) measuring glucose production by the second and the fourth samples of hepatic cells of step a) after incubation without the compound, comprising: iv) measuring glucose secreted by the hepatic cells in the second sample, thereby obtaining a second measurement; v) measuring glucose secreted by the hepatic cells in the fourth sample, thereby obtaining a fourth measurement; vi) subtracting the fourth measurement from the second measurement to obtain a second value of hepatic glucose production from gluconeogenesis after incubation of the hepatic cells without the compound; and(d) comparing the first value obtained in step (b) with the second value obtained in step (c), wherein if the first value obtained in step (b) is greater than the second value obtained in step (c), the compound is identified to increase gluconeogenesis in hepatic cells, and wherein if the first value obtained in step (b) is less than the second value obtained in step (c), the compound is identified to decrease gluconeogenesis in hepatic cells.

3. The method of claim 1, wherein if the first measurement is greater than the second measurement, the compound is indicated to increase hepatic glucose production.

4. The method of claim 1, wherein there is no difference between the first measurement and the second measurement, the compound is indicated to not affect hepatic glucose production, and wherein if the first measurement is less than the second measurement, the compound is indicated to decrease hepatic glucose production.

5. A method of identifying hepatic cells that have increased or decreased hepatic glucose production in response to treatment with a compound, the method comprising:(a) incubating a fifth and a sixth sample of hepatic cells in glucose production media, wherein the fifth sample comprises the compound and wherein the sixth sample does not comprise the compound;(b) measuring glucose secreted by the hepatic cell in the fifth sample, thereby obtaining a fifth measurement;(c) measuring glucose secreted by hepatic cells in the sixth sample, thereby obtaining a sixth measurement;(d) comparing the fifth measurement and the sixth measurement,wherein if the fifth measurement is greater than the sixth measurement, the hepatic cells are identified to have increased hepatic glucose production in response to treatment with the compound, and wherein if the fifth measurement is less than the sixth measurement, the hepatic cells are identified to have decreased hepatic glucose production in response to treatment with the compound.

6. The method of any one of claims 1 to 5, wherein the compound is a stimulatory agent or an inhibitory agent.

7. The method of any one of claims 1 to 6, wherein the compound is an inhibitory agent, and wherein the inhibitory agent is a hypoglycaemic compound.

8. The method of any one of claims 1 to 7, wherein the compound is an inhibitory agent, and wherein the inhibitory agent is selected from the group consisting of insulin, metformin, glyburide and pioglitazonc.

9. The method of any one of claims 1 to 6, wherein the compound is a stimulatory agent, and wherein the stimulatory agent is selected from the group consisting of forskolin, dexamethasone, dibutyryl-cyclic AMP, and 3-isobutyl-l- methylxanthine (IBMX).

10. The method of any one of the preceding claims, wherein the hepatic cells are selected from the group consisting of primary hepatocytes, liver organoids, HepG2 cells, human hepatic cancer cells, human pluripotent stem cell (hPSC) -derived hcpatocytc-likc cells, human pluripotent stem cell (hPSC)-derived hepatocytes, and human pluripotent stem cell (hPSC)-derived liver organoids, and optionally wherein the hepatic cells are genetically modified.

11. The method of any one of the preceding claims, wherein one or more compounds are tested simultaneously.

12. The method of any one of the preceding cells, wherein the incubating step is between 5 to 8 hours, or 6 hours.

13. The method of any one of the preceding claims, wherein the method further comprises normalizing the amount of glucose measured in each sample to a total protein content obtained from the same sample.

14. The method of any one of the preceding claims, wherein the samples are serum-starved overnight before step (a).

15. The method of claim 13, wherein the samples are washed with a buffer to remove residual glucose after being serum- starved and before step (a).

16. The method of any one of the preceding claims, wherein the glucose production media does not comprise glucose, serum, phenol red, pyruvate, or a combination thereof.

17. The method of any one of the preceding claims, wherein the gluconeogenic substrate is sodium pyruvate, sodium lactate, or a combination thereof.

18. The method of any one of the preceding claims, wherein the amount of glucose is measured fluorometrically, photometrically, colorimetrically, or spectrophotometrically.

19. A kit comprising a glucose production media, a buffer to wash cells, a cell lysis buffer, one or more solutions that couples proteins to a quantifiable tag, and instruction manual to measure hepatic glucose production from gluconeogenesis and / or glycogenolysis.

20. A kit comprising a glucose production media, one or more stimulatory or inhibitory agents, a buffer to wash cells, a cell lysis buffer, one or more solutions that couples proteins to a quantifiable tag, and instruction manual to determine whether hepatic glucose production from gluconeogenesis and / or glycogenolysis is increased or decreased under stimulatory or inhibitory conditions.

21. The kit of claim 21, for use in a method of identifying a compound that increases or decreases hepatic glucose production.

22. The kit of claim 21 or 22, for use in a method of identifying hepatic cells that have increased or decreased hepatic glucose production in response to treatment with a stimulatory agent or an inhibitory agent.

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