WO2017062363A1

WO2017062363A1 - Methods of use of betatrophin

Info

Publication number: WO2017062363A1
Application number: PCT/US2016/055332
Authority: WO
Inventors: Peng YI; Mary Elizabeth Patti; Pei-shan CAI
Original assignee: Joslin Diabetes Center
Priority date: 2015-10-05
Filing date: 2016-10-04
Publication date: 2017-04-13

Abstract

Methods and compositions for lowering blood glucose in subjects having hyperglycemia and diabetes are encompassed. Also encompassed are compositions and methods for increasing blood glucose in hypoglycemia, such as, for example, after gastric bypass surgery.

Description

METHODS OF USE OF BETATROPHIN

DESCRIPTION CROSS REFERENCE PARAGRAPH

[001] This application claims priority to United States provisional application number US62/237,374, filed October 5, 2015, and the entirety of the contents are incorporated by reference herein.

FIELD

[002] This application relates to betatrophin and agents that modulate

betatrophin (also known as Angptl8, RIFL and lipasin) for use in treatment of

hyperglycemia or hypoglycemia.

BACKGROUND

[003] Abnormal glucose levels can result from a number of diseases and physical conditions. For example, Type 1 and Type 2 diabetes mellitus result in hyperglycemia (increased levels of glucose). The American Diabetes Association (ADA) estimates the prevalence of diabetes mellitus at 29.1 million Americans, or 9.3% of the population (see ADA Statistics About Diabetes, 2015). Diabetes mellitus is estimated to be the seventh leading cause of death in the United States. Abnormally high glucose levels can also lead to atherosclerosis, kidney disease, stroke, nerve damage, and blindness. Pregnant women and their unborn children are at particular risk of adverse events caused by high glucose levels. In contrast to diabetes mellitus, hypoglycemia (low blood sugar) can be associated with treatments such as insulin administration or gastric bypass surgery and can lead to confusion, loss of consciousness, seizures, and death (see Rabiee J Surg Res. 2011 May 15; 167(2): 199-205).

[004] A number of factors regulate blood glucose levels. Insulin is released from beta cells located in the islets of the pancreas. Insulin is a hormone that works to reduce high blood glucose levels by stimulating cells of the body to uptake glucose from the blood and promote fat storage. Normally, blood glucose levels are tightly controlled by the beta cells, which release insulin to remove excess glucose from the blood.

[005] In Type 1 diabetes mellitus, an autoimmune response causes an individual's own immune system to attack and destroy beta cells. Type 2 diabetes, which is much more prevalent than Type 1 diabetes mellitus, is a condition where beta cells may be able to secrete insulin, but the cells of the body have developed insulin resistance and a diminished response to insulin. In both Type 1 and Type 2 diabetes mellitus,

pharmacologic interventions, such as administration of insulin, may be needed to control blood glucose levels. However, presently available treatments for diabetes do not provide the same degree of glycemic control as functional pancreatic beta cells, and diabetes mellitus is associated with a number of comorbid conditions, such as hypertension, dyslipidemia, heart attack, stroke, blindness, kidney disease, and amputations (see ADA Statistics About Diabetes, 2015).

[006] Both Type 1 and Type 2 diabetes mellitus lead to large reductions in beta cell mass (see Matveyenko and Butler, Diabetes Obes Metab 10(4): 23-31 (2008)). It has been proposed that treatments that replenish beta cell mass in diabetic subjects would allow long-term control of glycemic levels. While beta cells can expand rapidly during embryonic and neonatal periods, beta cells normally replicate at an extremely low rate in adults (see Yi et al Cell 153:747-758 (2013)).

[007] In vivo blockade of insulin signaling leads to compensatory expansion of beta cells and an increase in insulin secretion (see Yi 2013). In studying mechanisms of the response to insulin receptor antagonism, betatrophin was identified in mice as a hormone that is enriched in the liver and fat (see Yi 2013). It was reported that hydrodynamic injection of betatrophin expression constructs to the liver led to increased beta cell replication, and a concomitant lowering of fasting blood glucose levels, and improved glucose tolerance at seven days after administration of the plasmid. It has also been reported that stem cells expressing betatrophin began to decrease blood glucose levels three days after administration in rats with strepto2ocin (STZ)-induced diabetes (see CN104164451).

[008] However, further reports with knockout mice found no evidence that betatrophin plays a role in glucose homeostasis, instead implicating betatrophin in triglyceride metabolism (see Wang et al., PNAS 110: 40 16109-16114 (2013)). In fact, published reports indicated that neither knockout nor overexpression of betatrophin affect glucose metabolism (see Kaestner, Cell Metab 20(6): 932-933 (2014)). Thus, currently there is no common understanding of the role of betatrophin in glucose homeostasis. SUMMARY

[009] We have discovered that betatrophin has an acute glucose-lowering effect within 1-3 hours after administration. Betatrophin has a previously unreported ability to reduce blood glucose that is independent of beta cell replication. Binding partners for betatrophin, which can be targets for regulating betatrophin effects, are also described. Increased levels of betatrophin were found in a subpopulation of subjects reported to be hypoglycemic post-gastric bypass. Inhibition of betatrophin may be administered to these subjects to reduce betatrophin levels and regulate glucose status and ameliorate hypoglycemia.

[0010] In some embodiments, a method of lowering blood glucose in a subject comprising administering betatrophin is encompassed. Blood glucose levels may be lowered by about 72, 48, 24, 12, 6, 5, 4, 3, 2, or 1 hour(s) after administration of betatrophin.

[0011] In some embodiments, the subject has increased levels of insulin. In some embodiments, the subject's insulin levels are not increased. In some embodiments, blood glucose is measured using a glucose tolerance test. In some embodiments, glucose uptake is increased in liver, which can be detected by measuring plasma L-lactate levels. In some embodiments, glucose uptake is increased in brown adipose tissue (BAT) or heart.

[0012] In other embodiments, a method of treating diabetes mellitus in a subject comprising administering betatrophin is encompassed. In some embodiments, blood glucose levels are lowered by about 72, 48, 24, 12, 6, 5, 4, 3, 2, or 1 hour(s) after administration of betatrophin. The diabetes mellitus may be Type I or Type II.

[0013] In another embodiment, a method of increasing insulin levels in a subject comprising administering betatrophin is encompassed. Insulin levels in the blood may be increased by about 72, 48, 24, 12, 6, 5, 4, 3, 2, or 1 hour(s) after administration.

[0014] Further, a method of increasing the sensitivity of cells to insulin in a subject comprising administering betatrophin is encompassed, wherein in one

embodiment, the insulin sensitivity of the cells of the body are increased by about 72, 48, 24, 12, 6, 5, 4, 3, 2, or 1 hour(s) after administration.

[0015] A method of decreasing the excretion of glucose from the liver in a subject comprising administering betatrophin is also encompassed. In one embodiment the decrease in the excretion of glucose from the liver into the blood is seen by about 72, 48, 24, 12, 6, 5, 4, 3, 2, or 1 hour(s) after administration.

[0016] A method of treating hyperglycemia in a subject comprising administering betatrophin is described. In one embodiment the hyperglycemia is considered treated when blood glucose levels are less than 200 mg/ dl within about 72, 48, 24, 12, 6, 5, 4, 3, 2, or 1 hour(s) after administration of betatrophin.

[0017] In some embodiments, in each of the methods described herein, such as, for example, the treatment of hyperglycemia, lowering of blood glucose, increasing insulin levels, increasing sensitivity of cells to insulin, and decreasing the excretion of glucose from the liver is independent of beta cell replication or proliferation. Being "independent of beta cell replication or proliferation" means that beta cell replication or proliferation is not responsible for the physiologic effect. One can determine that beta cells are not responsible for the physiologic effect by determining the timing of the effect. If the physiological effect is seen prior to one week, then the physiological effect is considered to be independent of beta cell proliferation or replication.

[0018] In some embodiments, the subject has Type I diabetes mellitus or Type II diabetes mellitus. In one embodiment, the subject has a blood sugar level higher than 11.1 mmol/liter or 200 mg/ dl.

[0019] The subject in any of the methods described herein is a mammal. In one embodiment the mammal is a human, non-human primate, cow, horse, pig, sheep, goat, dog, cat, or rodent.

[0020] In some embodiments, betatrophin is administered at a dose of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000

[0021] In some embodiments, the betatrophin is administered in combination with an additional treatment. For example, betatrophin may be administered together with insulin, such as, for example, rapid-acting, intermediate-acting, or long-acting insulin.

[0022] In other embodiments, the additional agent to be administered in combination with betatrophin is a glucagon-like peptide analog or agonist, dipeptidyl peptidase-4 inhibitor, amylin analog, biguanide, thia2olidinedione, sulfonylurea, meglitinide, alpha-glucosidase inhibitor, or sodium/ glucose transporter 2 inhibitor. [0023] In other embodiments, a functional fragment of betatrophin is administered. The fragment may be any fragment that results in the desired physiological effect. In one embodiment the fragment comprises amino acids 22 to 76 of SEQ ID NO: 1, ammo acids 48-76 of SEQ ID NO: 1, or ammo acids 77 to 135 of SEQ ID NO: 1.

[0024] Betatrophin may be administered in a complex with a lipoprotein, such as, for example, high density lipoprotein (HDL) or a low density lipoprotein (LDL).

[0025] In some embodiments, methods of decreasing blood glucose in a subject are encompassed, wherein an agonist for a receptor that binds betatrophin is

administered. In one embodiment, the receptor for betatrophin is MARCO, RTN4R, hemopexin, or Asgrl. The agonist may be, for example, a small molecule, protein, or peptide.

[0026] Also encompassed are methods of increasing blood glucose in a subject comprising administering a betatrophin inhibitor, wherein the inhibitor blocks signaling of a betatrophin receptor. As non-limiting examples, MARCO, RTN4R, hemopexin, and Asgrl are betatrophin receptors. The inhibitors may be an antibody, decoy receptor, small molecule, protein, or peptide. In some embodiments, the betatrophin inhibitor is a muscarinic receptor antagonist. In some embodiments, the muscarinic receptor antagonist is atropine.

[0027] In some embodiments, the subject in need of increasing blood glucose has hypoglycemia. In one embodiment, the hypoglycemic subject is hypoglycemic post bariatric surgery. The bariatric surgery may be gastric banding or gastric bypass. In one embodiment, the gastric bypass surgery is Roux-en-Y gastric bypass.

[0028] Also encompassed is a method of lowering blood glucose in a subject comprising administering betatrophin and HDL, a method of increasing insulin levels in a subject comprising administering betatrophin and HDL, a method of increasing the sensitivity of cells to insulin in a subject comprising administering betatrophin and HDL, and a method of decreasing the excretion of glucose from the liver in a subject comprising administering betatrophin and HDL.

[0029] A method of treating hyperglycemia in a subject comprising administering betatrophin and HDL, wherein hyperglycemia is treated when blood glucose levels are less than 200 mg/ dl is encompassed. [0030] In each of the methods described herein, when betatrophin and HDL are used, the treatment of hyperglycemia, lowering of blood glucose, increasing insulin levels, increasing sensitivity of cells to insulin, and decreasing the excretion of glucose from the liver may independent or dependent of beta cell proliferation or replication.

[0031] In methods where the effect is independent of beta cell proliferation or replication, the effect may be seen within about 72, 48, 24, 12, 6, 5, 4, 3, 2, or 1 hour(s) after administration of betatrophin/HDL.

[0032] In methods where the effect is dependent on beta cell proliferation or replication, the effect may be seen no earlier than about 72 hours.

[0033] Additional objects and advantages will be set forth in part in the

description which follows, and in part will be obvious from the description, or may be learned by practice. The objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

[0034] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims.

[0035] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and together with the

description, serve to explain the principles described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figures 1A, IB, and 1C show results with maltose binding protein (MBP) conjugated recombinant betatrophin constructs. Figure 1 A shows a Commassie blue staining of MBP, MBP conjugated mouse betatrophin (MBP-mBT), and MBP conjugated human betatrophin (MBP-hBT). MBP or MBP-hBT was administered by i.v. injection for two days at 5Λμg/ day to CD1 mice. Figure IB shows pancreatic staining of Ki67 (a marker of cell proliferation) and insulin (a marker of beta cells) in mice at Day 3. The scale bar in Figure IB represents ΙΟΟμΜ. Figure 1C provides quantification of beta cell proliferation measured as the percentage of Ki67⁺-positive cells per insulin-positive cells (Ki67⁺/Insulin⁺) in the pancreatic slices. ***=P<0.001.

[0037] Figures 2A, 2B, and 2C show the dose-response of MBP-hBT on beta cell replication and the effects of betatrophin peptide administration on blood glucose levels. MBP or MBP-hBT were administered at 0.688, 1.375, 2.75, 5.5, 11, or 22ng/day for two days. Data on the beta cell replication (percentage of Ki67⁺/Insulin⁺ cells) are presented in Figure 2A. Figure 2B presents data on blood glucose measurements

(mg/ dL) taken at 0, 1, 3, 6, and 24 hours after a single injection of MBP (n=8) or MBP- hBT (n=8) into random-fed mice. Intraperitoneal glucose tolerance test [IPGTT] results are presented in Figure 2C for mice treated for two days with MBP (n=8) or MBP-hBT (n=8).

[0038] Figure 3 shows immunoblotting results for Angptl proteins following blue native polyacrylamide gel electrophoresis (BN-PAGE) from human serum samples. Human serum samples were run on BN-PAGE followed by immunoblotting with antibodies specific for Angptll-7, betatrophin (Angptl8), and markers of HDL particles (ApoAl and ApoE).

[0039] Figures 4A, 4B, 4C, 4D, and 4E show data on the presence of betatrophin and other Angptl proteins in high-density lipoprotein (HDL) particles. Figure 4A shows immunoblotting results following blue native polyacrylamide gel

electrophoresis (BN-PAGE) from human serum samples. Immunoblots were incubated with antibodies against betatrophin, AngptB, or Angptl4. The right blot of Figure 4A shows immunoblotting results following BN-PAGE of mouse serum after hydrodynamic tail vein injection of an expression vector that expresses myc-tagged mouse betatrophin protein. Figure 4B shows cholesterol levels following fractionation of postprandial human serum by gel filtration chromatography. Figure 4C shows expression of ApoAl and ApoE (HDL markers) and Angptll-7 proteins in fractions of human serum. Figure 4D shows cholesterol fractionation results of mouse serum measured two days after administration of MBP or MBP-hBT by hydrodynamic tail vein injection. Figure 4E shows detection of ApoAl and recombinant betatrophin protein in fractions of mouse serum after administration of MBP-hBT.

[0040] Figures 5A, 5B and 5C show the effect of lipid removal agent (LRA) on the presence of HDL markers and Angptl proteins in human serum and HDL. Human serum (shown in Figure 5A and 5B) and HDL fraction (shown in Figure 5C) were prepared with or without LRA and then analy2ed by western blot for HDL markers (ApoAl or ApoE) or Angptl proteins. [0041] Figures 6A and 6B show results from immunoprecipitation of human HDL with antibodies against Angptll-7 and betatrophin and results from fractionation of human serum on an ApoAl FPLC column. Figure 6A shows the results of immunoblots with antibodies against ApoAl and ApoE following immunoprecipitation from HDL using specific Angptl and betatrophin antibodies. Figure 6B shows immunoblot results for samples that were bound and then eluted from an ApoE FPLC column compared with the non- ApoAl FPLC fraction, i.e. the portion of HDL that did not bind to the ApoE column.

[0042] Figure 7 shows beta cell proliferation results in recipient mice receiving serum from donor mice treated with S961, an insulin antagonist, or from donor mice treated with PBS. Donor mice were treated with lOnM of S961 or PBS for 7 days. Serum and concentrated HDL fractions were prepared from the donor mice, and then 400μΕ of serum or HDL fraction from S961- or PBS-treated donor mice were administered to recipient mice (n=3 for each treatment group). One day after administration of serum or HDL fraction to recipient mice, pancreatic slices were prepared and measurements made of beta cell proliferation (Ki67⁺/Insulin⁺ %).

[0043] Figures 8A, 8B, 8C, 8D, and 8E show screening data for betatrophin receptors. Figure 8A presents a schematic of screening of expression libraries with an alkaline phosphate coupled human betatrophin (AP-hBT) followed by reaction with BCIP/NBT. Figure 8B shows native human serum and denatured human serum samples run on BN-PAGE gels followed by immunoblotting for human betatrophin. Figure 8B also shows results with mouse serum from an animal injected with MBP-hBT with native and denatured samples run on BN-PAGE gels followed by immunoblotting for human betatrophin. Figure 8C shows results of cells expressing GFP, MARCO, or RTN4R that were incubated with AP-hBT and stained for alkaline phosphatase using BCIP/NBT. Figure 8D shows IPGTT results from 5-week old MARCO knockout (KO) mice. Figure 8E shows IPGTT results from 8-week old RTN4R mice; data are adapted from published results from the phenotype database Mutant Mouse Resource and Research Centers (MMRRC).

[0044] Figures 9A, 9B, 9C, and 9D show results on the interaction of cells expressing mouse Asgrl and hemopexin (HPX) with alkaline phosphatase coupled mouse betatrophin (AP-mBT) followed by BCIP/NBT. Figures 9A and 9B show that no AP signal was seen when GFP-expressing Cos cells were incubated with AP-mBT or when mAsgrl -expressing Cos cells were incubated with AP alone. As shown in Figure 9C, incubation of mAsgrl -expressing Cos cells with AP-mBT followed by BCIP/NBT led to characteristic precipitate. Figure 9D shows that precipitate was also seen following incubation of mHPX-expressing Cos cells with AP-mBT followed by BCIP/NBT.

[0045] Figures 10A, 10B, and IOC show results on islet size and serum

betatrophin levels in healthy controls and subjects who developed hypoglycemia following Roux-en-Y gastric bypass (RYGB), i.e., post-RYGB. Figure 10A shows islet size in control subjects and subjects who developed hypoglycemia post-RYGB. Figures 10B and IOC show two independent experiments assessing serum betatrophin levels in controls and subjects who developed hypoglycemia post-RYGB.

[0046] Figures 11A, 11B, 11C, 11D, HE, 11F, 11G, 11H, 111, 11J, UK, 11L, and 11M show results on short-term effects of in vivo administration of recombinant MBP- hBT. Figure 11 A shows Commas sie blue staining and immunoblotting (IB) with anti- MBP and anti-liBT antibodies using the MBP and MBP-hBT constructs prepared for in vivo experiments. Figure 11B shows the experimental design with serial sample collections over 24 hours. Figure 11C shows plasma glucose levels over 24 hours after

administration of l^g of either MBP or MBP-hBT to CD1 mice (n=13 per group). Figure 1 ID shows the delta of basal blood glucose at 3 hours after injection of a range of doses of MBP or MBP-hBT (n=3-5 per group). Figure HE shows the experimental design of experiments to study the effects of administration of l ^g of either MBP or MBP-hBT at 0, 1, 2, and 3 hours post-injection (n=4 per group). Measurements of blood glucose (11F), insulin (11G), glucagon (l lFT), L-lactate (HI), triglycerides (11J) or cholesterol (11K) are shown. Figure 11L shows the experimental design of 2- deoxyglucose (2-DG) experiments, and Figure 11M shows results of 2-DG uptake by different tissues following administration of MBP or MBP-hBT. ** indicates P<0.01, * indicates P<0.05.

[0047] Figures 12A, 12B, 12C, 12D, 12E, 12F, 12G, 12H, 121, and 12J show effects of recombinant MBP-hBT administration followed by glucose tolerance tests. Figure 12A shows experimental design of IPGTT experiment. Figure 12B shows blood glucose levels and Figure 12C shows area under the curve (AUC) during IPGTT; n=15. Figure 12D shows serum insulin levels during IPGTT; n=10. Figure 12E shows experimental design of oral glucose tolerance test (OGTT) experiment. Figure 12F shows blood glucose levels, and Figure 12G shows AUC during OGTT; n=5. Figure 12H shows serum insulin levels during OGTT; n=5. Figure 121 shows experimental design of OGTT with collection at 30 minutes by cardiac puncture. Figure 12J shows plasma L- lactate levels at 30 min time point of the OGTT procedure. * indicates P<0.05; ** indicates P<0.01; *** indicates P<0.001.

[0048] Figures 13A, 13B, 13C, and 13D show effects of atropine on the response to MBP-hBT. Figure 13A shows the effect of atropine on glucose following

administration of either MBP or MBP-hBT. Figure 13B shows the results of atropine on glucose levels over time, and Figure 13C shows results of atropine on AUC glucose levels in the IPGTT model following administration of either MBP or MBP-hBT. Figure 13D shows effects of atropine on insulin levels in the IPGTT model. * indicates P<0.05; ** indicates P<0.01; *** indicates P<0.001; # indicates P<0.05; ## indicates P<0.01; ### indicates P<0.001. *indicates comparisons of MBP-hBT to MBP, and # indicates comparison of MBP-hBT to MBP-hBT + atropine.

[0049] Figures 14A, 14B, 14C, and 14D show experiments with strepto2ocin (STZ) diabetic mice and injection of MBP-hBT. Figure 14A shows experimental design for generating and studying STZ mice. Figure 14B shows plasma insulin levels in STZ mice following administration of MBP or MBP-hBT. Figure 14C shows blood glucose over time, and Figure 14D shows delta of basal blood glucose over time following administration of MBP or MBP-hBT to STZ mice. * indicates P<0.05; ** indicates P<0.01.

DESCRIPTION OF THE SEQUENCES

Table 1 provides a listing of certain sequences referenced herein.

Glu Val Ala Gin Ala Gin Lys Val Leu Arg Asp Ser Val Gin Arg Leu Glu Val Gin Leu Arg Ser Ala Trp Leu Gly Pro Ala Tyr Arg Glu Phe Glu Val Leu Lys Ala His Ala Asp Lys Gin Ser His lie Leu Trp Ala Leu Thr Gly His Val Gin Arg Gin Arg Arg Glu Met Val Ala Gin Gin His Arg Leu Arg Gin lie Gin Glu Arg Leu His Thr Ala Ala Leu Pro Ala

Mouse Met Ala Val Leu Ala Leu Cys Leu Leu Trp Thr Leu Ala betatrophin Ser Ala Val Arg Pro Ala Pro Val Ala Pro Leu Gly Gly

Pro Glu Pro Ala Gin Tyr Glu Glu Leu Thr Leu Leu Phe His Gly Ala Leu Gin Leu Gly Gin Ala Leu Asn Gly Val Tyr Arg Ala Thr Glu Ala Arg Leu Thr Glu Ala Gly His Ser Leu Gly Leu Tyr Asp Arg Ala Leu Glu Phe Leu Gly Thr Glu Val Arg Gin Gly Gin Asp Ala Thr Gin Glu Leu Arg Thr Ser Leu Ser Glu He Gin Val Glu Glu Asp Ala Leu His Leu Arg Ala Glu Ala Thr Ala Arg Ser Leu Gly Glu Val Ala Arg Ala Gin Gin Ala Leu Arg Asp Thr Val Arg Arg Leu Gin Val Gin Leu Arg Gly Ala Trp Leu Gly Gin Ala His Gin Glu Phe Glu Thr Leu Lys Ala Arg Ala Asp Lys Gin Ser His Leu Leu Trp Ala Leu Thr Gly His Val Gin Arg Gin Gin Arg Glu Met Ala Glu Gin Gin Gin Trp Leu Arg Gin He Gin Gin Arg Leu His Thr Ala Ala Leu Pro Ala

Rat Met Val Val Pro He Leu Cys Leu Leu Trp Ala He Ala betatrophin Thr Ala Val Arg Pro Ala Pro Val Ala Pro Leu Gly Gly

Pro Glu Pro Ala Gin Tyr Glu Glu Leu Thr Leu Leu Phe His Gly Ala Leu Gin Leu Gly Gin Ala Leu Asn Gly Val Tyr Lys Ala Thr Glu Ala Arg Leu Thr Glu Ala Gly Arg Asn Leu Gly Leu Phe Asp Gin Ala Leu Glu Phe Leu Gly Arg Glu Val Asn Gin Gly Arg Asp Ala Thr Arg Glu Leu Arg Thr Ser Leu Ser Glu He Gin Ala Glu Glu Asp Thr Leu His Leu Arg Ala Glu Ala Thr Ala Arg Ser Leu Arg Glu Val Ala Arg Ala Gin His Ala Leu Arg Asn Ser Val Arg Arg Leu Gin Val Gin Leu Arg Gly Ala Trp Leu Gly Gin Ala His Gin Glu Phe Glu Asn Leu Lys Asp Arg Ala Asp Lys Gin Asn His Leu Leu Trp Ala Leu Thr Gly His Val Gin Arg Gin Gin Arg Glu Met Ala Glu Gin Gin Gin Trp Leu Arg Gin He Gin Gin Arg Leu His Met Ala Ala Leu Pro Ala

Consensus Met Xaai Val Pro Ala Leu Cys Leu Leu Trp Ala Leu Ala betatrophin Xaa₂ Ala Val Arg Pro Ala Pro Val Ala Pro Leu Gly Gly sequence Pro Glu Pro Ala Gin Tyr Glu Glu Leu Thr Leu Leu Phe

His Gly Ala Leu Gin Leu Gly Gin Ala Leu Asn Gly Val Tyr Arg Ala Thr Glu Ala Arg Leu Thr Glu Ala Gly Xaa₃ Ser Leu Gly Leu Tyr Asp Arg Ala Leu Glu Phe Leu Gly Xaa₄ Glu Val Xaa₅ Gin Gly Arg Asp Ala Thr Gin Glu Leu Arg Thr Ser Leu Ser Glu He Gin Xaa₆ Glu Glu Asp Xaa₇ Leu His Leu Arg Ala Glu Ala Thr Ala Arg Ser Leu Gly Glu Val Ala Arg Ala Gin Xaa₈ Ala Leu Arg Asp Ser Val Arg Arg Leu Gin Val Gin Leu Arg Gly Ala Trp Leu Gly Gin Ala His Gin Glu Phe Glu Xaa₉ Leu Lys Ala Arg Ala Asp Lys Gin Ser His Leu Leu Trp Ala Leu Thr Gly His Val Gin Arg Gin Gin Arg Glu Met Ala Glu Gin Gin Gin Trp Leu Arg Gin He Gin Gin Arg Leu His Thr Ala Ala Leu Pro Ala

Wherein Xaai is Pro, Ala or Val and

Wherein Xaa₂ is Met, Ser or Thr and

Wherein Xaa₃ is Asn, His or Arg and

Wherein Xaa₄ is Gin, Thr or Arg and

Wherein Xaas is Ser, Arg or Asn and Wherein Xaae is Met, Val or Ala and

Wherein Xaa₇ is lie, Ala or Thr and

Wherein Xaae is Lys, Gin or His and

Wherein Xaa₉ is Val, Thr or Asn

DNA sequence ATGAAAATCG AAGAAGGTAA ACTGGTAATC TGGATTAACG of Maltose GCGATAAAGG CTATAACGGT CTCGCTGAAG TCGGTAAGAA binding ATTCGAGAAA GATACCGGAA TTAAAGTCAC CGTTGAGCAT protein CCGGATAAAC TGGAAGAGAA ATTCCCACAG GTTGCGGCAA

(MBP) CTGGCGATGG CCCTGACATT ATCTTCTGGG CACACGACCG

CTTTGGTGGC TACGCTCAAT CTGGCCTGTT GGCTGAAATC ACCCCGGACA AAGCGTTCCA GGACAAGCTG TATCCGTTTA CCTGGGATGC CGTACGTTAC AACGGCAAGC TGATTGCTTA CCCGATCGCT GTTGAAGCGT TATCGCTGAT TTATAACAAA GATCTGCTGC CGAACCCGCC AAAAACCTGG GAAGAGATCC CGGCGCTGGA TAAAGAACTG AAAGCGAAAG GTAAGAGCGC GCTGATGTTC AACCTGCAAG AACCGTACTT CACCTGGCCG CTGATTGCTG CTGACGGGGG TTATGCGTTC AAGTATGAAA ACGGCAAGTA CGACATTAAA GACGTGGGCG TGGATAACGC TGGCGCGAAA GCGGGTCTGA CCTTCCTGGT TGACCTGATT AAAAACAAAC ACATGAATGC AGACACCGAT TACTCCATCG CAGAAGCTGC CTTTAATAAA GGCGAAACAG CGATGACCAT CAACGGCCCG TGGGCATGGT CCAACATCGA CACCAGCAAA GTGAATTATG GTGTAACGGT ACTGCCGACC TTCAAGGGTC AACCATCCAA ACCGTTCGTT GGCGTGCTGA GCGCAGGTAT TAACGCCGCC AGTCCGAACA AAGAGCTGGC AAAAGAGTTC CTCGAAAACT ATCTGCTGAC TGATGAAGGT CTGGAAGCGG TTAATAAAGA CAAACCGCTG GGTGCCGTAG CGCTGAAGTC TTACGAGGAA GAGTTGGTGA AAGATCCGCG TATTGCCGCC ACTATGGAAA ACGCCCAGAA AGGTGAAATC ATGCCGAACA TCCCGCAGAT GTCCGCTTTC TGGTATGCCG TGCGTACTGC GGTGATCAAC GCCGCCAGCG GTCGTCAGAC TGTCGATGAA GCCCTGAAAG ACGCGCAGAC TAATTCGAGC TCGAACAACA ACAACAATAA CAATAACAAC AACCTCGGGA TCGAGGGAAG GATTTCACAT ATGTCCATGG GCGGCCGCGA TATCGTCGAC GGATCCGAAT TCCCTGCAGG TAATTAA

Protein Met Lys He Glu Glu Gly Lys Leu Val He Trp He Asn sequence Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys

MBP Lys Phe Glu Lys Asp Thr Gly He Lys Val Thr Val Glu

His Pro Asp Lys Leu Glu Glu Lys Phe Pro Gin Val Ala Ala Thr Gly Asp Gly Pro Asp He He Phe Trp Ala His Asp Arg Phe Gly Gly Tyr Ala Gin Ser Gly Leu Leu Ala Glu He Thr Pro Asp Lys Ala Phe Gin Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys Leu He Ala Tyr Pro He Ala Val Glu Ala Leu Ser Leu He Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu Glu He Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn Leu Gin Glu Pro Tyr Phe Thr Trp Pro Leu He Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr Asp He Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val Asp Leu He Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr Ser He Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala Met Thr He Asn Gly Pro Trp Ala Trp Ser Asn He Asp Thr Ser Lys Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gin Pro Ser Lys Pro Phe Val Gly Val Leu Ser Ala Gly He Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu Leu Val Lys Asp Pro Arg He Ala Ala Thr Met Glu Asn Ala Gin Lys Gly Glu He Met Pro Asn He Pro Gin Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val lie Asn Ala Ala Ser Gly Arg Gin Thr Val Asp Glu Ala Leu Lys Asp Ala Gin Thr Asn Ser Ser Ser Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Leu Gly lie Glu Gly Arg lie Ser His Met Ser Met Gly Gly Arg Asp lie Val Asp Gly Ser Glu Phe Pro Ala Gly Asn

DNA sequence ATGAAAATCG AAGAAGGTAA ACTGGTAATC TGGATTAACG of MBP-mouse GCGATAAAGG CTATAACGGT CTCGCTGAAG TCGGTAAGAA betatrophin ATTCGAGAAA GATACCGGAA TTAAAGTCAC CGTTGAGCAT

(MBP-mBT) CCGGATAAAC TGGAAGAGAA ATTCCCACAG GTTGCGGCAA

CTGGCGATGG CCCTGACATT ATCTTCTGGG CACACGACCG CTTTGGTGGC TACGCTCAAT CTGGCCTGTT GGCTGAAATC ACCCCGGACA AAGCGTTCCA GGACAAGCTG TATCCGTTTA CCTGGGATGC CGTACGTTAC AACGGCAAGC TGATTGCTTA CCCGATCGCT GTTGAAGCGT TATCGCTGAT TTATAACAAA GATCTGCTGC CGAACCCGCC AAAAACCTGG GAAGAGATCC CGGCGCTGGA TAAAGAACTG AAAGCGAAAG GTAAGAGCGC GCTGATGTTC AACCTGCAAG AACCGTACTT CACCTGGCCG CTGATTGCTG CTGACGGGGG TTATGCGTTC AAGTATGAAA ACGGCAAGTA CGACATTAAA GACGTGGGCG TGGATAACGC TGGCGCGAAA GCGGGTCTGA CCTTCCTGGT TGACCTGATT AAAAACAAAC ACATGAATGC AGACACCGAT TACTCCATCG CAGAAGCTGC CTTTAATAAA GGCGAAACAG CGATGACCAT CAACGGCCCG TGGGCATGGT CCAACATCGA CACCAGCAAA GTGAATTATG GTGTAACGGT ACTGCCGACC TTCAAGGGTC AACCATCCAA ACCGTTCGTT GGCGTGCTGA GCGCAGGTAT TAACGCCGCC AGTCCGAACA AAGAGCTGGC AAAAGAGTTC CTCGAAAACT ATCTGCTGAC TGATGAAGGT CTGGAAGCGG TTAATAAAGA CAAACCGCTG GGTGCCGTAG CGCTGAAGTC TTACGAGGAA GAGTTGGTGA AAGATCCGCG TATTGCCGCC ACTATGGAAA ACGCCCAGAA AGGTGAAATC ATGCCGAACA TCCCGCAGAT GTCCGCTTTC TGGTATGCCG TGCGTACTGC GGTGATCAAC GCCGCCAGCG GTCGTCAGAC TGTCGATGAA GCCCTGAAAG ACGCGCAGAC TAATTCGAGC TCGAACAACA ACAACAATAA CAATAACAAC AACCTCGGGA TCGAGGGAAG GATTTCACAT ATGTCCATGG GCGGCCGCGA TATCGTCGAC GGATCCGAAT TCCCTGCAGG TAATGAAAAC CTATACTTCC AATCAGGAGC CCCCATGGGC GGCCCAGAAC TGGCACAGCA TGAGGAGCTG ACCCTGCTCT TCCATGGGAC CCTGCAGCTG GGCCAGGCCC TCAACGGTGT GTACAGGACC ACGGAGGGAC GGCTGACAAA GGCCAGGAAC AGCCTGGGTC TCTATGGCCG CACAATAGAA CTCCTGGGGC AGGAGGTCAG CCGGGGCCGG GATGCAGCCC AGGAACTTCG GGCAAGCCTG TTGGAGACTC AGATGGAGGA GGATATTCTG CAGCTGCAGG CAGAGGCCAC AGCTGAGGTG CTGGGGGAGG TGGCCCAGGC ACAGAAGGTG CTACGGGACA GCGTGCAGCG GCTAGAAGTC CAGCTGAGGA GCGCCTGGCT GGGCCCTGCC TACCGAGAAT TTGAGGTCTT AAAGGCTCAC GCTGACAAGC AGAGCCACAT CCTATGGGCC CTCACAGGCC ACGTGCAGCG GCAGAGGCGG GAGATGGTGG CACAGCAGCA TCGGCTGCGA CAGATCCAGG AGAGACTCCA CACAGCGGCG CTCCCAGCCT GA

MBP-mBT Lys Phe Glu Lys Asp Thr Gly He Lys Val Thr Val Glu

His Pro Asp Lys Leu Glu Glu Lys Phe Pro Gin Val Ala Ala Thr Gly Asp Gly Pro Asp He He Phe Trp Ala His Asp Arg Phe Gly Gly Tyr Ala Gin Ser Gly Leu Leu Ala Glu He Thr Pro Asp Lys Ala Phe Gin Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys Leu He Ala Tyr Pro He Ala Val Glu Ala Leu Ser Leu He Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu Glu He Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn Leu Gin Glu Pro Tyr Phe Thr Trp Pro Leu He Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr Asp He Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val Asp Leu He Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr Ser He Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala Met Thr He Asn Gly Pro Trp Ala Trp Ser Asn He Asp Thr Ser Lys Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gin Pro Ser Lys Pro Phe Val Gly Val Leu Ser Ala Gly He Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu Leu Val Lys Asp Pro Arg He Ala Ala Thr Met Glu Asn Ala Gin Lys Gly Glu He Met Pro Asn He Pro Gin Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val He Asn Ala Ala Ser Gly Arg Gin Thr Val Asp Glu Ala Leu Lys Asp Ala Gin Thr Asn Ser Ser Ser Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Leu Gly He Glu Gly Arg He Ser His Met Ser Met Gly Gly Arg Asp He Val Asp Gly Ser Glu Phe Pro Ala Gly Asn Glu Asn Leu Tyr Phe Gin Ser Gly Ala Pro Val Ala Pro Leu Gly Gly Pro Glu Pro Ala Gin Tyr Glu Glu Leu Thr Leu Leu Phe His Gly Ala Leu Gin Leu Gly Gin Ala Leu Asn Gly Val Tyr Arg Ala Thr Glu Ala Arg Leu Thr Glu Ala Gly His Ser Leu Gly Leu Tyr Asp Arg Ala Leu Glu Phe Leu Gly Thr Glu Val Arg Gin Gly Gin Asp Ala Thr Gin Glu Leu Arg Thr Ser Leu Ser Glu He Gin Val Glu Glu Asp Ala Leu His Leu Arg Ala Glu Ala Thr Ala Arg Ser Leu Gly Glu Val Ala Arg Ala Gin Gin Ala Leu Arg Asp Thr Val Arg Arg Leu Gin Val Gin Leu Arg Gly Ala Trp Leu Gly Gin Ala His Gin Glu Phe Glu Thr Leu Lys Ala Arg Ala Asp Lys Gin Ser His Leu Leu Trp Ala Leu Thr Gly His Val Gin Arg Gin Gin Arg Glu Met Ala Glu Gin Gin Gin Trp Leu Arg Gin He Gin Gin Arg Leu His Thr Ala Ala Leu Pro Ala

DNA sequence ATGAAAATCG AAGAAGGTAA ACTGGTAATC TGGATTAACG of MBP-human GCGATAAAGG CTATAACGGT CTCGCTGAAG TCGGTAAGAA betatrophin ATTCGAGAAA GATACCGGAA TTAAAGTCAC CGTTGAGCAT

(MBP-hBT) CCGGATAAAC TGGAAGAGAA ATTCCCACAG GTTGCGGCAA

CTGGCGATGG CCCTGACATT ATCTTCTGGG CACACGACCG CTTTGGTGGC TACGCTCAAT CTGGCCTGTT GGCTGAAATC ACCCCGGACA AAGCGTTCCA GGACAAGCTG TATCCGTTTA CCTGGGATGC CGTACGTTAC AACGGCAAGC TGATTGCTTA CCCGATCGCT GTTGAAGCGT TATCGCTGAT TTATAACAAA GATCTGCTGC CGAACCCGCC AAAAACCTGG GAAGAGATCC CGGCGCTGGA TAAAGAACTG AAAGCGAAAG GTAAGAGCGC GCTGATGTTC AACCTGCAAG AACCGTACTT CACCTGGCCG CTGATTGCTG CTGACGGGGG TTATGCGTTC AAGTATGAAA ACGGCAAGTA CGACATTAAA GACGTGGGCG TGGATAACGC TGGCGCGAAA GCGGGTCTGA CCTTCCTGGT TGACCTGATT AAAAACAAAC ACATGAATGC AGACACCGAT TACTCCATCG CAGAAGCTGC CTTTAATAAA GGCGAAACAG CGATGACCAT CAACGGCCCG TGGGCATGGT CCAACATCGA CACCAGCAAA GTGAATTATG GTGTAACGGT ACTGCCGACC TTCAAGGGTC AACCATCCAA ACCGTTCGTT GGCGTGCTGA GCGCAGGTAT TAACGCCGCC AGTCCGAACA AAGAGCTGGC AAAAGAGTTC CTCGAAAACT ATCTGCTGAC TGATGAAGGT CTGGAAGCGG TTAATAAAGA CAAACCGCTG GGTGCCGTAG CGCTGAAGTC TTACGAGGAA GAGTTGGTGA AAGATCCGCG TATTGCCGCC ACTATGGAAA ACGCCCAGAA AGGTGAAATC ATGCCGAACA TCCCGCAGAT GTCCGCTTTC TGGTATGCCG TGCGTACTGC

GGTGATCAAC GCCGCCAGCG GTCGTCAGAC TGTCGATGAA GCCCTGAAAG ACGCGCAGAC TAATTCGAGC TCGAACAACA ACAACAATAA CAATAACAAC AACCTCGGGA TCGAGGGAAG GATTTCACAT ATGTCCATGG GCGGCCGCGA TATCGTCGAC GGATCCGAAT TCCCTGCAGG TAATGAAAAC CTATACTTCC AATCAGGAGC CCCCATGGGC GGCCCAGAAC TGGCACAGCA TGAGGAGCTG ACCCTGCTCT TCCATGGGAC CCTGCAGCTG GGCCAGGCCC TCAACGGTGT GTACAGGACC ACGGAGGGAC GGCTGACAAA GGCCAGGAAC AGCCTGGGTC TCTATGGCCG CACAATAGAA CTCCTGGGGC AGGAGGTCAG CCGGGGCCGG GATGCAGCCC AGGAACTTCG GGCAAGCCTG TTGGAGACTC AGATGGAGGA GGATATTCTG CAGCTGCAGG CAGAGGCCAC AGCTGAGGTG CTGGGGGAGG TGGCCCAGGC ACAGAAGGTG CTACGGGACA GCGTGCAGCG GCTAGAAGTC CAGCTGAGGA GCGCCTGGCT GGGCCCTGCC TACCGAGAAT TTGAGGTCTT AAAGGCTCAC GCTGACAAGC AGAGCCACAT CCTATGGGCC CTCACAGGCC ACGTGCAGCG GCAGAGGCGG GAGATGGTGG CACAGCAGCA TCGGCTGCGA CAGATCCAGG AGAGACTCCA CACAGCGGCG CTCCCAGCCT GA

MBP-hBT Lys Phe Glu Lys Asp Thr Gly He Lys Val Thr Val Glu

His Pro Asp Lys Leu Glu Glu Lys Phe Pro Gin Val Ala Ala Thr Gly Asp Gly Pro Asp He He Phe Trp Ala His Asp Arg Phe Gly Gly Tyr Ala Gin Ser Gly Leu Leu Ala Glu He Thr Pro Asp Lys Ala Phe Gin Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys Leu He Ala Tyr Pro He Ala Val Glu Ala Leu Ser Leu He Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu Glu He Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn Leu Gin Glu Pro Tyr Phe Thr Trp Pro Leu He Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr Asp He Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val Asp Leu He Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr Ser He Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala Met Thr He Asn Gly Pro Trp Ala Trp Ser Asn He Asp Thr Ser Lys Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gin Pro Ser Lys Pro Phe Val Gly Val Leu Ser Ala Gly He Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu Leu Val Lys Asp Pro Arg He Ala Ala Thr Met Glu Asn Ala Gin Lys Gly Glu He Met Pro Asn He Pro Gin Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val He Asn Ala Ala Ser Gly Arg Gin Thr Val Asp Glu Ala Leu Lys Asp Ala Gin Thr Asn Ser Ser Ser Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Leu Gly He Glu Gly Arg He Ser His Met Ser Met Gly Gly Arg Asp He Val Asp Gly Ser Glu Phe Pro Ala Gly Asn Glu Asn Leu Tyr Phe Gin Ser Gly Ala Pro Met Gly Gly Pro Glu Leu Ala Gin His Glu Glu Leu Thr Leu Leu Phe His Gly Thr Leu Gin Leu Gly Gin Ala Leu Asn Gly Val Tyr Arg Thr Thr Glu Gly Arg Leu Thr Lys Ala Arg Asn Ser Leu Gly Leu Tyr Gly Arg Thr He Glu Leu Leu Gly Gin Glu Val Ser Arg Gly Arg Asp Ala Ala Gin Glu Leu Arg Ala Ser Leu Leu Glu Thr Gin Met Glu Glu Asp He Leu Gin Leu Gin Ala Glu Ala Thr Ala Glu Val Leu Gly Glu Val Ala Gin Ala Gin Lys Val Leu Arg Asp Ser Val Gin Arg Leu Glu Val Gin Leu Arg

I. Definitions

[0051] In addition to definitions included in this sub-section, further definitions of terms are interspersed throughout the text.

[0052] In this invention, "a" or "an" means "at least one" or "one or more," etc., unless clearly indicated otherwise by context. The term "or" means "and/ or" unless stated otherwise. In the case of a multiple-dependent claim, however, use of the term "or" refers back to more than one preceding claim in the alternative only. [0053] As used herein, betatrophin includes full length and functional fragments of betatrophin, as well as variations at non-conserved amino acids that maintain blood glucose lowering function.

[0054] The term "treatment," as used herein, covers any administration or application of a therapeutic for disease in a subject, and includes inhibiting the disease, arresting its development, relieving one or more symptoms of the disease, or preventing reoccurrence of one or more symptoms of the disease. For example, treatment of diabetes type I and type II subjects may comprise alleviating hyperglycemia intermittently such that a subject is hyperglycemic, and upon administration of betatrophin is less hyperglycemic or non-hyperglycemic as compared to a time point prior to administration.

[0055] As used herein, "beta cell independent" and the like is used herein to describe a physiological effect that is not dependent on beta cell proliferation or replication. Whether or not the blood glucose regulation is independent of beta cells can be determined by assessing the time from administration to effect. If the effect is caused by beta cells, the effect would not be seen until about one week after administration. Therefore, an effect in less than one week is considered "beta cell independent."

II. Betatrophin Compositions

[0056] Betatrophin is a protein also known as Angptl8, TD26, RIFL, PROH 85, and PVPA599. Betatrophin has been shown to be released in conditions where insulin signaling is blocked by the insulin antagonist S961 (see Yi 2013). S961 binds to the insulin receptor and antagonizes insulin signaling. In in vivo models, administration of S961 leads to an upregulation of beta cell proliferation and also to release of betatrophin from liver and fat.

[0057] Administration of vectors expressing betatrophin (see Yi 2013) or stem cells expressing betatrophin (see CN104164451) indicate an ability of betatrophin to stimulate beta cell replication. Delivery of gene plasmids expressing betatrophin by ultrasound- targeted microbubble destruction (UTMD, see Chen et al, Diabetologia 58:1036-1044 (2015)) also leads to an increase in beta cell replication.

[0058] Results have shown a delayed effect of betatrophin overexpression. For example, in Yi 2013, changes in glucose tolerance tests were measured in mice six days after injection of plasmid. Using UTMD for delivery of betatrophin-expressing plasmids, it was shown that fasting blood glucose levels were improved in treated diabetic rats starting only after seven or more days following delivery (see Chen 2015). Thus, the effects of betatrophin on beta cell replication and glucose levels have been characterized to have a delayed time to onset. We herein show for the first time that betatrophin also has a fast-acting effect on blood glucose levels that cannot be attributed to beta cell proliferation or regeneration.

[0059] Sequences for human (SEQ ID NO: 1), mouse (SEQ ID NO: 2), and rat (SEQ ID NO: 3) betatrophm have been described (see US20140303078). In addition, a consensus sequence for betatrophin has been described, based on amino acid differences between species (SEQ ID NO: 4). A number of functional domains of betatrophin also have been described, including a coiled-coil domain (see US20140303078). As used within this application, "betatrophin" in inclusive of the full-length protein, the protein lacking its native signal sequence, or any other functional fragment of the protein.

[0060] In some embodiments, the betatrophin administered is recombinant betatrophin. In some embodiments, the betatrophin administered to a subject comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In some embodiments, the betatrophin is full-length betatrophin. In some embodiments, the betatrophin is a polypeptide that is smaller than full-length betatrophin. In some embodiments, a functional fragment of betatrophin is administered. In some

embodiments, the functional fragment of betatrophin is betatrophin lacking its native signal sequence. In some embodiments, the functional fragment of betatrophin comprises the amino acid sequence of amino acids 22-76, 48-76, or 77-135 of SEQ ID NO: 1. Variations within full length and less than full length fragments are encompassed so long as the betatrophin retains functional activity, i.e., retains the ability to lower blood glucose levels in hyperglycemic subjects after administration to a subject within at least 72 hours post administration.

1. Agents that promote betatrophin signaling

[0061] Betatrophin signaling may be promoted in a number of ways. In some embodiments, recombinant betatrophin is administered. In some embodiments, an agonist for a receptor for betatrophin is administered. In some embodiments, the receptor is MARCO, RTN4R, hemopexin, or Asgrl . In some embodiments, the agonist for the receptor is a small molecule, protein, or peptide. In each of the methods described herein that comprise administering of betatrophin, it is understood that an agent that is an agonist for a receptor of betatrophin is also encompassed.

2. Agents that inhibit betatrophin signaling

[0062] Betatrophin signaling may be inhibited in a number of ways. In some embodiments, an inhibitor of betatrophin signaling is administered. In some

embodiments, the betatrophin inhibitor is an antibody that binds betatrophin. In some embodiments, a betatrophin inhibitor is an agent that is an antagonist for a receptor for betatrophin. In some embodiments, this receptor is MARCO, RTN4R, hemopexin, or Asgrl. In some embodiments, the inhibitor is an antibody, decoy receptor, small molecule, protein, or peptide. In some embodiments, an inhibitor of betatrophin signaling decreases beta cell proliferation or replication.

[0063] In some embodiments, the inhibitor of betatrophin signaling is an inhibitor of muscarinic cholinergic receptors. In some embodiments, the inhibitor of betatrophin signaling is atropine. In some embodiments, the inhibitor of betatrophin signaling is an inhibitor of muscarinic cholinergic receptors that regulate insulin release from pancreatic beta cells.

[0064] In some embodiments, atropine inhibits the ability of betatrophin to decrease blood glucose. In some embodiments, atropine inhibits the ability of

betatrophin to decrease blood glucose in response to a glucose tolerance test. In some embodiments, atropine inhibits the effect of betatrophin to increase insulin release in response to a glucose tolerance test.

III. Methods of Treatment

[0065] In each embodiment of the invention, the subject treated is a mammal. In one embodiment, the mammal is a human, non-human primate, cow, horse, pig, sheep, goat, dog, cat, or rodent. In embodiment, the subject is a human subject.

[0066] Glucose levels in the blood are normally tightly regulated to maintain an appropriate source of energy for cells of the body. Dysregulation of blood sugar must be ameliorated to maintain health and longevity, and therapies that are fast acting are especially desired. Such fast acting therapies allow subjects to monitor blood glucose in real time and immediately self-medicate themselves to bring glucose levels within normal limits. Dosing with exogenous insulin is one example of a fast-acting glucose modulator that has allowed subjects with diabetes to maintain relatively normal lifestyles. Described herein is a non-insulin fast-acting compound that regulates blood glucose levels in realtime.

[0067] Insulin and glucagon are principal hormones that regulate blood glucose levels. In response to an increase in blood glucose, such as after a meal, insulin is released from beta cells of the pancreas. Insulin regulates the metabolism of carbohydrates and fats by promoting uptake of glucose from the blood into fat and skeletal muscle. Insulin also promotes fat storage and inhibits the release of glucose by the liver. Regulation of insulin levels is a primary means for the body to regulate glucose in the blood.

[0068] When glucose levels in the blood are decreased, insulin is no longer released and instead glucagon is released from the alpha cells of the pancreas. Glucagon causes the liver to convert stored glycogen into glucose and to release this glucose into the bloodstream. Thus, insulin and glucagon work in concert to regulate blood glucose levels.

[0069] We herein describe novel and nonobvious compositions and methods to regulate abnormal blood glucose. In one embodiment, betatrophin is administered to a subject to lower blood glucose, wherein the lowering of blood glucose occurs in less time than it would take to regenerate beta cells. For example, blood glucose is lowered in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 48, or 72 hours after administration of betatrophin. In one embodiment, the blood glucose level is lowered in 1-3, 3-6, 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27, 27-30, 30-33, 33-36, 36- 39, 39-41, 41-44, 44-47, 47-50, 50-53, 53-56, 56-59, 59-61, 61-63, 63-66, 66-69, or 69-72 hours after administration of betatrophin.

[0070] Conversely, in one embodiment, a betatrophin inhibitor is administered to a subject to raise blood glucose levels, wherein the raising of blood glucose occurs in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 48, or 72 hours after administration of betatrophin inhibitor. In one embodiment, the blood glucose level is raised in 1-3, 3-6, 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27, 27-30, 30-33, 33-36, 36- 39, 39-41, 41-44, 44-47, 47-50, 50-53, 53-56, 56-59, 59-61, 61-63, 63-66, 66-69, or 69-72 hours after administration of betatrophin inhibitor.

[0071] Hyperglycemia refers to an increased levels of glucose in the blood.

Hyperglycemia can be associated with high levels of sugar in the urine, frequent urination, and increased thirst. Diabetes mellitus refers to a medical state of hyperglycemia.

[0072] The American Diabetes Association (ADA) suggests that FPG levels of lOOmg/ dL to 125 mg/ dL or HbAlc levels of 5.7% to 6.4% may be considered hyperglycemia and may indicate that a subject is at high risk of developing diabetes mellitus (i.e. prediabetes, see ADA Guidelines 2015).

[0073] The ADA states that a diagnosis of diabetes mellitus may be made in a number of ways. A diagnosis of diabetes mellitus can be made in a subject displaying an HbAlc level of≥6.5%, an FPG levels of≥126mg/ dL, a 2-hour plasma glucose of ≥200mg/ dL during an OGTT, or a random plasma glucose level≥200mg/ dL in a subject with classic symptoms of hyperglycemia.

[0074] Diabetes mellitus can be broken into Type 1 and Type 2. Type 1 diabetes mellitus (previously known as insulin-dependent diabetes or juvenile diabetes) is an autoimmune disease characterized by destruction of the insulin-producing beta cells of the pancreas. Classic symptoms of Type 1 diabetes mellitus are frequent urination, increased thirst, increased hunger, and weight loss. Subjects with Type 1 diabetes mellitus are dependent on administration of insulin for survival.

[0075] Type 2 diabetes mellitus is a metabolic disease characterized by a relative decrease in insulin levels and/ or a phenotype of insulin resistance. Insulin resistance refers to when cells of the body no longer respond appropriately to insulin. The risk of Type 2 diabetes mellitus is increased in individuals who are obese or who have a sedentary lifestyle.

[0076] While exercise, dietary changes, and weight loss may be able to alleviate Type 2 diabetes in some subjects, many subjects need pharmacologic treatment to control blood glucose levels. Exogenous insulin may be administered for treatment of Type 2 diabetes with rapid-acting, intermediate-acting, and long-acting insulins available. Other non-insulin injectable medications may be used to treat Type 2 diabetes, including glucagon-like peptide analogs and agonists, dipeptidyl peptidase-4 inhibitors, and amylin analogs. Oral medications may be used to treat Type 2 diabetes, including biguanides, thiazolidinediones, sulfonylureas, meglitinides, alpha-glucosidase inhibitors, and sodium/glucose transporter 2 inhibitors. [0077] In the absence of regulation of glucose levels in subjects with diabetes, a range of serious complications may be seen. These include atherosclerosis, kidney disease, stroke, nerve damage, and blindness.

[0078] In some embodiments, a subject is administered betatrophin at a dose of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000μ¾/1¾. [

[0079] In some embodiments, a subject is administered betatrophin to lower blood glucose. In some embodiments, blood glucose is lowered by about 72, 48, 24, 12, 6, 5, 4, 3, 2, or 1 hour(s) after administration.

[0080] In some embodiments, a method of increasing insulin levels is

encompassed comprising administering betatrophin. In some embodiments, the increase in insulin levels occurs about 72, 48, 24, 12, 6, 5, 4, 3, 2, or 1 hour(s) after administration of betatrophin.

[0081] In some embodiments, a method of increasing insulin sensitivity of the cells of the body is encompassed comprising administering betatrophin. In some embodiments, the increase in insulin sensitivity occurs about 72, 48, 24, 12, 6, 5, 4, 3, 2, or 1 hour(s) after administration of betatrophin.

[0082] In some embodiments, a method of increasing glucose uptake by liver is encompassed comprising administering betatrophin. In some embodiments, the increase in glucose uptake by liver occurs about 72, 48, 24, 12, 6, 5, 4, 3, 2, or 1 hour(s) after administration of betatrophin. In some embodiments, glucose uptake by the liver is measured by an increase in plasma L-lactate. In some embodiments, the increase in plasma L-lactate occurs about 72, 48, 24, 12, 6, 5, 4, 3, 2, or 1 hour(s) after administration of betatrophin. In some embodiments, glucose uptake is increased by brown adipose tissue (BAT) heart following administration of betatrophin. In some embodiments, glucose uptake is increased by heart following administration of betatrophin.

[0083] In some embodiments, a method of decreasing the excretion of glucose from the liver is encompassed comprising administering betatrophin. In some

embodiments, the decrease in excretion of glucose from the liver occurs about 72, 48, 24, 12, 6, 5, 4, 3, 2, or 1 hour(s) after administration of betatrophin. [0084] In each of the described methods of lowering of blood glucose in a subject administered betatrophin, the lowering of blood glucose is fast-acting (e.g., less than about 72 hours), and is therefore independent of beta cell replication/proliferation.

[0085] In some embodiments, administering betatrophin causes a decrease in blood glucose levels such that levels are less than 200 mg/ dL. In some embodiments, this decrease in blood glucose levels such that levels are less than 200 mg/ dL occurs within about 72, 48, 24, 12, 6, 5, 4, 3, 2, or 1 hour(s) after administration of betatrophin.

[0086] In some embodiments, the effect of administering betatrophin to decrease blood glucose levels is measured by a glucose tolerance test.

[0087] In some embodiments, administering betatrophin decreases blood glucose levels after an oral glucose tolerance test. In some embodiments, administering betatrophin decreases blood glucose levels at 5, 10, 15, 30, 45, 60, 90, or 120 minutes after an oral glucose tolerance test.

[0088] In some embodiments, administering betatrophin increases insulin levels after an oral glucose tolerance test. In some embodiments, administering betatrophin increases insulin levels at 5, 10, 15, 30, 45, 60, 90, or 120 minutes after an oral glucose tolerance test.

A. Methods of treating hyperglycemia

[0089] In some embodiments, the invention comprises methods of treating hyperglycemia comprising administering betatrophin. In some aspects, methods of treating hyperglycemia comprising administering betatrophin to a hyperglycemic subject is encompassed, wherein blood glucose levels are reduced to below about 200 mg/ dL, 150 mg/dL, lOOmg/dL, or about 125 mg/dL in less than 72, 48, 24, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 hour(s) after administration of betatrophin are encompassed.

[0090] In some embodiments, a method for regulating blood glucose is encompassed comprising administering betatrophin to a hyperglycemic subject. In one embodiment, a method for regulating blood glucose is encompassed comprising administering betatrophin to a hyperglycemic subject, wherein blood glucose levels are reduced to below about 200 mg/ dL, 150 mg/ dL, lOOmg/ dL, or about 125 mg/ dL in less than 72, 48, 24, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 hour(s) after administration of betatrophin. [0091] In some embodiments, a method of treating hyperglycemia is encompassed comprising administering betatrophin to a subject having blood glucose levels at about 200 mg/ dL, 150 mg/ dL, lOOmg/ dL, or about 125 mg/ dL, wherein blood glucose is lowered to less than 200 mg/ dL, 150 mg/ dL, lOOmg/ dL, or about 125 mg/ dL in less than about 72 hours after administration of betatrophin. In one embodiment, blood glucose is lowered in less than about 48, 24, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 hour(s) post administration with betatrophin.

[0092] In some embodiments, the subject treated with betatrophin has

hyperglycemia based on diagnostic criteria of the American Diabetes Association. In some embodiments, the subject has FPG levels of lOOmg/ dL to 125 mg/ dL. In some embodiments, the subject has HbAlc levels of 5.7% to 6.4%. In some embodiments, the subject with hyperglycemia has prediabetes.

[0093] In some embodiments, hyperglycemia is deemed treated when blood glucose levels are less than 200mg/ dl. In some embodiments, hyperglycemia is deemed treated when blood glucose levels are less than 200mg/ dl within about 72, 48, 24, 12, 6, 5, 4, 3, 2, or 1 hour(s) after administration of betatrophin. In some embodiments, hyperglycemia is treated when blood glucose levels are less than 200mg/ dl, and this effect is independent of beta cell replication or proliferation.

B. Methods of treating diabetes mellitus

[0094] A method of treating diabetes mellitus comprising administering

betatrophin is encompassed. In one embodiment, the method comprises lowering blood glucose levels in the diabetic subject to below about 200 mg/ dL, 150 mg/ dL, lOOmg/ dL, or about 125 mg/dL in less than 72, 48, 24, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 hour(s) after administration of betatrophin. In one embodiment, blood glucose is lowered in less than about 48, 24, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 hour(s) post administration with betatrophin.

[0095] In some embodiments, the subject treated with betatrophin has Type 1 diabetes mellitus. In some embodiments, the subject treated with betatrophin has Type 2 diabetes mellitus. In some embodiments, the diabetic subject treated with betatrophin has a relative decrease in insulin levels. In some embodiments, the subject treated with betatrophin has a phenotype of insulin resistance. In some embodiments, the subject treated with betatrophin has decreased beta cell mass. In some embodiments, the decrease in beta cell mass in a subject is due to an autoimmune disease.

[0096] In some embodiments, the subject treated with betatrophin has diabetes mellitus based on diagnosis criteria of the American Diabetes Association. In some embodiments, the subject with diabetes mellitus has an HbAlc level of≥6.5%. In some embodiments, the subject with diabetes mellitus has an FPG levels of≥126mg/ dL. In some embodiments, the subject with diabetes mellitus has a 2-hour plasma glucose of >200mg/ dL during an OGTT. In some embodiments, the subject with diabetes mellitus has a random plasma glucose level≥200mg/ dL or 11.1 mmol/L. In some embodiments, the subject with diabetes mellitus has a random plasma glucose level≥200mg/ dL or 11.1 mmol/L with classic symptoms of hyperglycemia.

[0097] In some embodiments, the subject treated with betatrophin has an increased release of insulin following administration of betatrophin. In some

embodiments, the subject treated with betatrophin does not have an increased release of insulin following administration of betatrophin.

[0098] In some embodiments, methods of treatment of diabetes mellitus by administering betatrophin are developed using animal models. In some embodiments, methods of treatment of diabetes mellitus by administering of betatrophin are developed using rodent models. In some embodiments, methods of treatment of diabetes mellitus by administering betatrophin are developed using the strepto2ocin (STZ) mouse model. In some embodiments, the STZ mouse model may comprise a single dose of STZ or multiple doses. In some embodiments, the STZ mouse model may comprise doses of 30- 80mg/kg for 3-5 days. In some embodiments, the STZ mouse model may comprise a single dose of 100-300 mg/kg is used. In some embodiments, the STZ mouse model is one wherein mice are treated with a single dose of 150 mg/kg STZ. In some

embodiments, methods of treatment of diabetes mellitus by administering of betatrophin are developed using the STZ mouse model, wherein mice used for testing are those that develop blood glucose levels of greater than 350mg/ dl at 2-3 days after treatment with STZ.

C. Methods of treating metabolic syndrome

[0099] Metabolic syndrome is the presence of a group of risk factors including high blood pressure, high blood sugar, abnormal cholesterol levels, and abdominal fat that significantly increase the risk of heart disease and diabetes mellitus. Diagnosis of the metabolic syndrome is based on the presence of a number of these specific risk factors in the same patient. For example, patients having three or more of the following risk factors may be diagnosed with metabolic syndrome: presence of high waist circumference, high triglycerides or use of a cholesterol-lowering medicine, low HDL or use of a cholesterol- lowering medicine, blood pressure greater than 135/ 85 or use of a blood pressure- treating medicine, and fasting blood glucose levels of lOOmg/ dl or higher. Along with lifestyle modification, pharmacologic treatment for metabolic syndrome (including treatment for improved glycemic and blood pressure measures and controlling

cholesterol) are appropriate to decrease risk of developing heart disease and diabetes mellitus.

[00100] A method of treating metabolic syndrome comprising administering betatrophin is encompassed. In one embodiment, the method comprises lowering blood glucose levels in the metabolic syndrome subject to below about 200 mg/ dL, 150 mg/ dL, lOOmg/dL, or about 125 mg/dL in less than 72, 48, 24, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 hour(s) after administration of betatrophin. In one embodiment, blood glucose is lowered in less than about 48, 24, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 hour(s) post administration with betatrophin.

D. Methods of treating hypoglycemia

[00101] Hypoglycemia describes a condition of abnormally low blood glucose. Hypoglycemia is often charactered as a blood glucose level of less than

70mg/dL. Signs of hypoglycemia include shakiness, nervousness, sweating, and rapid heart-rate. Hypoglycemia can cause sei2ures, unconsciousness, and death if left untreated.

[00102] In subjects being treated with insulin, hypoglycemia is a serious complication of delivery of too large of a dose of insulin. In these subjects, hypoglycemia may be referred to as an insulin reaction or insulin shock.

[00103] Hypoglycemia may also be seen in subjects following bariatric surgery, such as gastric bypass (see Rabiee 2011). Roux-en-Y gastric bypass (RYGB) surgery is a type of bariatric surgery where hypoglycemia has been often reported as a complication post-surgery. Hypoglycemia may occur at any time, but often, one to three years after RYGB surgery and is commonly thought to be due to expansion of beta cells in the pancreas. [00104] In one embodiment, a method for regulating blood glucose is encompassed comprising administering a betatrophin inhibitor to a hypoglycemic subject. In some embodiments, a method for regulating blood glucose is encompassed

comprising administering a betatrophin inhibitor to a hypoglycemic subject, wherein blood glucose levels are raised in said subject to greater than about 70 mg/ dL, 80 mg/ dL, 90 mg/ dL, or about 100 mg/ dL in less than 72, 48, or 24 hours after administration of betatrophin inhibitor.

[00105] In some embodiments, a method of treating hypoglycemia is envisioned comprising administering a betatrophin inhibitor to a subject having blood glucose levels lower than about 70 mg/ dL. In some embodiments, a method of treating hypoglycemia is envisioned comprising administering a betatrophin inhibitor to a subject having blood glucose levels lower than about 70 mg/ dL, wherein blood glucose is raised to more than about 70 mg/ dL in less than 72 hours after administration of betatrophin.

[00106] In some embodiments, a method for regulating blood glucose is encompassed comprising administering betatrophin inhibitor. In some embodiments, a method for regulating blood glucose is encompassed comprising administering

betatrophin inhibitor to a hypoglycemic subject. In some embodiments, blood glucose levels are increased to below about 200 mg/ dL, 150 mg/ dL, lOOmg/ dL, or about 125 mg/dL in less than about 72, 48, 24, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 hour(s) after administration of betatrophin inhibitor.

[00107] In some embodiments, a method of treating hypoglycemia is envisioned comprising administering betatrophin inhibitor to a subject having blood glucose levels below about 80mg/ dL. In some embodiments, a method of treating hypoglycemia is envisioned comprising administering betatrophin inhibitor to a subject having blood glucose levels below about 80mg/ dL, wherein blood glucose is raised to no greater than about lOOmg/ dL in less than 72 hours after administration of betatrophin. In one embodiment, blood glucose is raised in less than about 48, 24, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 hour(s) post administration of betatrophin inhibitor.

[00108] In some embodiments, the betatrophin inhibitor is an antibody that binds betatrophin. In some embodiments, the betatrophin inhibitor is an antibody that binds betatrophin and neutrali2es it glucose lowering activity. In some embodiments, the betatrophin inhibitor is an antagonist of a receptor for betatrophin. In some embodiments, this receptor is MARCO, RTN4R, hemopexin, or Asgrl. In some embodiments, the inhibitor is an antibody, decoy receptor, small molecule, protein, or peptide. In some embodiments, an inhibitor of betatrophin signaling decreases beta cell proliferation or replication. In some embodiments, an inhibitor of betatrophin signaling has no effect on beta cell proliferation or replication.

1. Hypoglycemia Following Bariatric Surgery

[00109] In some embodiments, a method of treating a subpopulation of post-bariatric surgery subjects who are hypoglycemic comprising administering a betatrophin inhibitor is encompassed. In some embodiments, the bariatric surgery is gastric banding. In some embodiments, the bariatric surgery is gastric bypass. In some embodiments, hypoglycemia occurs one to three years after gastric bypass surgery. In some embodiments, hypoglycemia in a subject following gastric bypass surgery is associated with expansion of beta cells. In some embodiments, the gastric bypass surgery is Roux-en-Y gastric bypass (RYGB) surgery.

[00110] In one embodiment a method of treating hypoglycemia post gastric bypass is encompassed comprising administering a betatrophin inhibitor.

IV. Association of Betatrophin with HDL

[00111] We herein report that native betatrophin is complexed with lipoprotein. Therefore, in each of the method of treatment embodiments described herein, betatrophin may be administered in complex with a lipoprotein. In some embodiments, the lipoprotein is a high density lipoprotein (HDL) or a low density lipoprotein (LDL).

[00112] In some embodiments, betatrophin and lipoprotein, such as HDL, are administered as a method of lowering blood glucose. In some embodiments, administering betatrophin and lipoprotein increases insulin levels, increases sensitivity of cells to insulin, or decreases excretion of glucose from the liver. In some embodiments, a method of treating hyperglycemia is described comprising administering betatrophin and lipoprotein, such as HDL. In some embodiments, a method of treating Type I and/ or Type II diabetes is described comprising administering betatrophin and lipoprotein, such as HDL. The subject is considered treated when the subject is no longer hyperglycemic. In some embodiments, a subject is no longer hyperglycemic when blood glucose levels are less than about 200 mg/ dL, about 150 mg/ dL, or about 125 mg/ dL. In some embodiments, effects of administering betatrophin and lipoprotein are independent of beta cell proliferation or replication. In some embodiments, effects of administering betatrophin and lipoprotein are dependent on beta cell proliferation or replication. In some embodiments, administering betatrophin and lipoprotein increases insulin levels, increases sensitivity of cells to insulin, or decreases excretion of glucose from the liver within about 72, 48, 24, 12, 6, 5, 4, 3, 2, or 1 hours(s) after administration. In some embodiments, administering betatrophin and lipoprotein increases insulin levels, increases sensitivity of cells to insulin, or decreases excretion of glucose from the liver only after at least 72 hours.

[00113] In some embodiments, betatrophin and lipoprotein, such as HDL, are administered as a method of increasing beta cell replication and/ or proliferation.

V. Measurements of Blood Glucose

[00114] A variety of tests can be used to measure blood glucose levels. The fasting plasma glucose (FPG) test measures blood glucose after a subject has not eaten for at least eight hours. A random blood sugar test measures blood glucose regardless of when a subject has eaten. An oral glucose tolerance test (OGTT) is a series of

measurement of blood glucose levels after a subject drinks a liquid containing glucose. A hemoglobin Ale test (HbAlc) measures the glucose associated with hemoglobin (i.e. glycosylated hemoglobin) and is considered a measure of a subject's average blood sugar level for the past two to three months. In diabetic and pre-diabetic patients, blood glucose is typically measured daily or multiple times a day with a home blood glucose monitor that measures the amount of glucose in a small amount of blood from a prick of the finger, for example.

[00115] Any of the above or other methods known in the art to test blood glucose levels may be used in any of the methods of the invention. In one embodiment, a first measurement of blood glucose is taken, and if the measurement is above normal as compared to a control with normal blood glucose levels, betatrophin is administered. Betatrophin is fast-acting, and therefore glucose levels in the blood will be restored to within normal limits within at least 72 hours after administration.

VI. Combination Therapy with Betatrophin

[00116] In each of the above-described methods/uses, such as, for example, in treating a subject with hyperglycemia, such as diabetes mellitus, a combination therapy may be administered comprising betatrophin (or agent that promotes betatrophin signaling) and an additional therapeutic agent. In some embodiments, the additional therapeutic agent is insulin. In some embodiments, the additional therapeutic agent is rapid-acting insulin. In some embodiments, the additional therapeutic agent is

intermediate-acting insulin. In some embodiments, the additional therapeutic agent is long-acting insulin. In some embodiments, the additional therapeutic agent is a non- insulin injectable medication. In some embodiments, the non-insulin injectable medication is a glucagon-like peptide analog or agonist, dipeptidyl peptidase-4 inhibitor, or amylin analog. In some embodiments, the additional therapeutic agent is an oral medication. In some embodiments, the additional therapeutic agent is a biguanide, thia2olidinedione, sulfonylurea, meglitinide, alpha-glucosidase inhibitor, or

sodium/ glucose transporter 2 inhibitor.

[00117] Combination treatments may be achieved by way of the

simultaneous, sequential, or separate dosing of the individual components of the treatment. Where the administration is sequential or separate, the delay in administering the second component should not be such as to lose the beneficial effect of the combination.

VII. Administration

[00118] Betatrophin may be administered by any means known in the art to administer proteins of similar size and biochemical properties. In one embodiment, betatrophin is administered parenterally, orally, buccally, transdermally, via sonophoresis, or via inhalation. In some embodiments, parenteral administration is subcutaneous, intramuscular, intrasternal, or intravenous injection.

[00119] In one embodiment, betatrophin is formulated in tablets, capsules, powders, granules, lozenges, suppositories, reconstitutable powders, or liquid

preparations, such as oral or sterile parenteral solutions or suspensions.

[00120] Oral liquid preparations may be in the form of, for example, emulsions, syrups, or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel, hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, fractionated coconut oil, oily esters such as esters of glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid; and if desired conventional flavoring or coloring agents.

[00121] For parenteral administration, fluid unit dosage forms may be prepared utilizing betatrophin and a sterile vehicle, and, depending on the concentration used, can be either suspended or dissolved in the vehicle. In preparing solutions, betatrophin can be dissolved in water for injection and filter sterilized before filling into a suitable vial or ampoule and sealing. Adjuvants such as a local anesthetic, a preservative and buffering agents can be dissolved in the vehicle. To enhance the stability, betatrophin can be frozen after filling into the vial and the water removed under vacuum. Parenteral suspensions are prepared in substantially the same manner, except that betatrophin is suspended in the vehicle instead of being dissolved, and sterilization cannot be accomplished by filtration. Betatrophin can be sterilized by exposure to ethylene oxide before suspending in the sterile vehicle. Advantageously, a surfactant or wetting agent is included in the composition to facilitate uniform distribution.

[00122] Compositions may contain from 0.1% to 99% by weight, preferably from 10-60% by weight, of the active material, depending upon the method of administration.

[00123] Compositions may, if desired, be in the form of a pack

accompanied by written or printed instructions for use.

EXAMPLES

Example 1. In vivo effects of MBP conjugated human betatrophin

[00124] Betatrophin is a protein also known as Angptl8, TD26, RIFL,

PROH85, and PVPA599. In vivo, betatrophin is a secreted protein (see Yi 2013).

Expression of recombinant betatrophin in vitro is hampered by its low solubility. For example, betatrophin expressed in an E. Coli expression system with either a

hexahistidine or a glutathione S-transferase tag, resulted in production of protein in inclusion bodies, which could not be purified (data not shown). Additionally, betatrophin expressed in yeast and eukaryotic expression systems can be secreted, but precipitates after purification. [00125] In order to produce recombinant betatrophin for use in experiments, betatrophin protein from mouse and human was expressed in E. Coli with a maltose binding protein (MBP) tag. DNA and protein sequences for MBP are SEQ ID NO: 5 and SEQ ID NO: 6, respectively. DNA and protein sequences for MBP-mouse betatrophin (MBP-mBT) are SEQ ID NO: 7 and SEQ ID NO: 8, respectively. DNA and protein sequences for MBP-human betatrophin (MBP-hBT) are SEQ ID NO: 9 and SEQ ID NO: 10, respectively. E. Coli expression vectors (pMal-c5x) were generated for mouse betatrophin and human betatrophin that lacked the endogenous signal peptide. These correspond to amino acids 19-198 of mouse betatrophin (NM 001080940) and amino acids 22-198 of human betatrophin (NM 018687). The pMal-c5x vectors were designed to express an MBP tag fused to the N-terminal of the peptide. The ClearColi BL21 strain (NEB) was used, as it is an endotoxin-free E. coli expression system.

Expression of protein from the plasmids was induced with 0.4mM IPTG, and

supernatants were collected for analysis of secreted protein.

[00126] Figure 1A shows Commassie blue staining of MBP alone (MBP), mouse betatrophin conjugated to MBP (MBP-mBT), and human betatrophin conjugated to MBP (MBP-hBT). These data indicate that use of the MBP tag improves solubility of betatrophin, as full-length MBP-mBT and MBP-hBT can be secreted by the E. Coli, whereas little to no secretion was seen when expression was tried without the MBP tag.

[00127] In addition to the full-length MBP-mBT and MBP-hBT, an additional band was seen for each of these constructs at approximately 37kDa (see Figure 1A). This construct is likely an MBP fragment that is generated by non-specific cleavage at the tobacco etch virus (TEV) -cleavage sites that are contained at the junction of the MBP and betatrophin proteins.

[00128] In vivo effects of recombinant betatrophin were then investigated.

MBP and MBP-hBT were injected into CD1 mice through the tail vein for two consecutive days at a dose of

On Day 3, pancreatic sections were taken and prepared for staining. Pancreatic sections were blocked with 10% donkey serum and were incubated with primary antibodies against insulin (Dako, A0564) and Ki67 (Abeam, abl6667)), at 4°C overnight. Secondary antibodies were applied after washing the slides for 3 x 10 min in PBST. Secondary antibodies (Life Technology, a21207 and al l073) were used for 1 h incubation at room temperature. Slides were mounted with Dapi containing mounting media.

[00129] Staining was done for Ki67 and insulin to evaluate beta cell replication. Ki67 is a marker of cell proliferation, and insulin is a marker of pancreatic beta cells. Therefore, an increase in the ratio of cells positive for Ki67 versus cells positive for insulin (i.e., %Ki57⁺/Insulin⁺) indicates a relative increase in beta cell proliferation. Figure IB shows representative images of pancreatic sections co- immunostained with insulin and Ki67 from mice treated for two days with either MBP or MBP-hBT. Figure 1C summari2es quantification of the percentage of beta cell replication in both treatment groups (i.e., % Ki57⁺/Insulin⁺). These results show that two-day MBP-hBT treatment at 5Λμg/ day caused a significant increase in the percentage of beta cell replication compared with MBP treatment alone (P<0.001).

[00130] The dose-response of MBP-hBT was then investigated. Mice were treated with a range of doses of betatrophin (0.69 to 22μ/ day) for two days, and then beta cell replication was measured at Day 3. Figure 2A presents results of the

%Ki57⁺/Insulin⁺ over the doses tested, showing a dose-dependent effect of MBP-hBT on beta cell replication. The peak in beta cell replication with two days of treatment was at l^g/ day MBP-hBT. No dose-response effect was seen with injection of MBP alone.

[00131] Blood glucose levels were also evaluated at time points over 24 hours after a single injection of l ^g MBP or MBP-hBT. As shown in Figure 2B, an acute glucose-lowering effect was seen after i.v. injection of betatrophin. This effect for MBP-hBT was significant versus MBP at 3 hours (P<0.001). MBP-hBT also decreases blood glucose at lhr in separate experiments (data note shown). As effects of betatrophin on beta cell replication would be expected to take at least a week to affect blood glucose levels, the acute glucose-lowering effects of betatrophin indicate a previously undescribed effect of betatrophin on blood glucose homeostasis that is independent of beta cell replication.

[00132] In order to verify the acute glucose-lowering effects of betatrophin, intraperitoneal glucose tolerance test (IPGTT) results were compared for mice treated with once-daily i.v. injection of l ^g MBP (n=8) or MBP-hBT (n=8) for two days. After two days of dosing with either MBP or MBP-hBT, mice were fasted for 16 hours after which lg/kg glucose was administered by i.p. injection. As shown in Figure 2C, mice treated with MBP-hBT had improved glucose tolerance compared to mice treated with MBP alone. Based on intraperitoneal insulin tolerance test results, mice treated with MBP-hBT had normal insulin sensitivity without significant differences from mice treated with MBP alone (data not shown). Thus, these data support an acute ability of betatrophin to improve glucose tolerance that is apparent after only 2 days of treatment with betatrophin, which is prior to effects on glucose would be expected based on an increased insulin release due to beta cell replication.

Example 2. Composition of Betatrophin in Serum and Binding Partners

[00133] Betatrophin (also known as Angptl8) belongs to the angiopoietin- link protein (Angptl) family, and the composition of circulating betatrophin and other Angptl proteins was next investigated.

[00134] To investigate the native composition of betatrophin and other

Angptl proteins in human serum, blue native polyacrylamide gel electrophoresis (BN- PAGE) gels were run with samples of human serum. Figure 3 shows the presence of Angptll-7 and betatrophin in large protein complexes in human serum samples using immunoblot analysis following BN-PAGE. These protein complexes ran at similar molecular weight to protein complexes present in immunoblots with antibodies against ApoAl (Abeam, ab 52945) and ApoE (Abeam, ab 1906). These results suggested that Angptl proteins, including betatrophin, may be contained in the HDL fraction of human serum.

[00135] Figure 4A shows results of immunoblots of human serum for betatrophin, AngptB, and Angptl4. Additionally, the left blot of Figure 4A also shows recombinant human betatrophin (hbetatrophin). These results indicate that native betatrophin, AngptB, and Angptl4 exist in large multi-protein complexes in the serum much larger than the Angptl or betatrophin proteins themselves.

[00136] Following administration of a vector engineered to express myc- labeled recombinant mouse betatrophin by hydrodynamic tail vein injection,

immunoblotting of BN-PAGE gels indicated that myc staining (using anti-myc tag (HRP) antibody, Abeam ab 1326) was also found in large protein complexes, as shown in Figure 4A. DNA and protein sequences for myc tag are SEQ ID NO: 11 and SEQ ID NO: 12, respectively. DNA and protein sequences for myc-tagged mouse betatrophin are SEQ ID NO: 13 and SEQ ID NO: 14, respectively. These data show that exogenously administered betatrophin is able to associate into large protein complexes in a similar manner to native betatrophin.

[00137] Next, fractionation was performed to determine what fraction of serum contains the betatrophin complexes. Postprandial human serum was subjected to gel filtration chromatography using a Superose 6 column (GE Healthcare Life Sciences). Results on cholesterol levels in different serum fractions are presented in Figure 4B. Next, different fractions were run on western blots and immunoblotted with antibodies staining either HDL markers (ApoAl and ApoE) or Angptll -7, as shown in Figure 4C. Antibodies used were ApoAl, Abeam ab 52945; ApoE, Abeam abl906; Angptll, Abeam abl07091; Angptl4, Abeam abll5798; AngptB, Abeam abl25718; Betatrophin, Phoenix G-051-55; Angtpl-2, -5, -6, -7, AdipoGen, AG-25A-0068, AG-25A-0069, AG-25A-0030, and AG-25A-0050, respectively. Staining for ApoAl and ApoE was in the same fractions as staining for Angptl proteins. These data support the presence of Angptll -7 in HDL-containing fractions of human serum.

[00138] Mouse serum from animals injected once-daily for two days with either l^g MBP or MBP-hBT by tail vein injection was also analy2ed. As shown in

Figure 4D, the HDL fraction is shifted to a larger si2e in mice administered MBP-hBT versus those administered MBP, which may be partly due to the incorporation of MBP- hBT recombinant protein into HDL particles. Figure 4E shows results of serum fractions following injection of MBP-hBT using immunoblots with human betatrophin antibody (Phoenix anti-human Betatrophin purified IgG, G-051-55). The presence of betatrophin by western blot analysis in fractions with expression of ApoAl confirms that exogenously administered betatrophin is incorporated into serum HDL particles.

[00139] The effect of lipid reducing agent (LRA) to decrease the presence of

Angptl proteins and HDL markers was analy2ed in human serum and fractionated HDL. In the LRA experiments, 20μ1 human serum with 180μ1 PBS or 200μ1 HDL fraction were incubated with 14.4 mg LRA for 50 min at room temperature. After incubation, samples were centrifuged at 2200g for 2 min. The supernatant parts were used for western blot.

[00140] As shown in Figures 5A and 5B, LRA treatment of serum decreased levels of HDL markers (ApoAl and ApoE) as well as Angptll-7 and betatrophin. Levels of IgG, which are not normally associated with HDL particles, were not affected by treatment of serum with LRA. Figure 5C shows that LRA treatment of the HDL fraction also decreased levels of ApoAl, betatrophin, and Angptll, 3, 4, 5, and 6. These data confirm the association of betatrophin and other Angptl proteins with lipids in the HDL fraction.

[00141] To further understand the association of betatrophin and other

Angptl proteins with HDL particles, immunoprecipitations were performed with Angptl- specific antibodies, followed by western blot analysis and immunoblotting with ApoAl (Abeam ab 52945) and ApoE (Abeam abl906), proteins that are present in HDL. The immunoprecipitation procedure followed the instructions of Thermo Scientific Pierce Co-Immunoprecipitation kit, #26149. Figure 6A shows expression of ApoAl and ApoE in total HDL (HDL input) and immunoprecipitations with Angptl-specific antibodies. ApoAl was detectable in all immunoprecipitations except for a control sample of no IgG. ApoE was clearly detectable in AngptB and 4 immunoprecipitations. Thus, Angptl proteins associate with protein markers contained in HDL.

[00142] The association of Angptl was further investigated with a fast protein liquid chromatography (FPLC) column of the HDL marker protein ApoAl . Proteins that can bind ApoAl will bind to the column and can then be eluted, while proteins that do not bind to ApoAl will be present in the non- ApoAl FPLC fraction. Figure 6B shows that betatrophin and other Angptl proteins were associated with the eluate from the ApoAl column. There was little signal seen in the non- ApoAl FPLC fraction. These data further support the association of betatrophin and other Angptl proteins with HDL particles.

[00143] The ability of serum or HDL fraction of donor mice to stimulate beta cell proliferation in recipient mice was then examined. Donor mice were treated with S961, a peptide that binds the insulin receptor and blocks insulin signaling. Treatment with S961 can stimulate release of betatrophin from the liver and white fat and stimulate beta cell replication (see Yi 2013). The ability of serum samples from treated mice to be able to transfer this effect to naive mice was studied.

[00144] Donor mice were treated with ΙΟηΜ S961 or PBS for 7 days by

ALZET subcutaneous osmotic pump (model number 2001). At day 7, sera were collected by cardiac puncture for serum transfer injection or HDL fraction isolation. 400μ1 S961- or PBS-treated serum were fractionated by FPLC (fast protein liquid chromatography). Cholesterol assay was used for the measurement of lipoprotein distribution. All HDL fractions were concentrated back to 400μ1 volume by Amicon Ultra Centrifugal Filter Unit with Ultracel-10 membrane.

[00145] Serum and HDL fractions were then prepared from the donor mice. Then 400μΕ of serum or HDL fraction from S961- or PBS-treated donor mice were administered to recipient mice (n=3 for each treatment group). One day after administration of serum or HDL fraction to recipient mice, pancreatic slices were prepared and measurements made of beta cell proliferation (Ki67⁺/Insulin⁺ %) as described in Example 1. Figure 7 shows that recipient mice receiving serum or HDL from donor mice treated with S961 had higher beta cell proliferation than recipient mice receiving serum or HDL from donor mice treated with PBS. These data are consistent with a soluble factor in serum and HDL, such as betatrophin, that is present in donor mice treated with S961 and that is capable of stimulating beta cell proliferation in recipient mice naive to treatment with S961. The fact that the HDL fraction is capable to produce this effect supports the fact that an HDL-associated protein, such as

betatrophin, is the active agent or one of the group of active agents capable of inducing beta cell proliferation.

[00146] The HDL-bound form of betatrophin may therefore be the active form of betatrophin in the serum. Exogenously administered betatrophin can incorporate into HDL particles and thus should bind to native binding partners for betatrophin within HDL.

Example 3. Identification of Membrane Proteins that Bind Betatrophin

[00147] As the signaling pathways activated by betatrophin are not clear, an unbiased search for receptors that bind betatrophin may uncover novel proteins or protein complexes that regulate islet cell homeostasis and/ or lipid or glucose metabolism.

[00148] Expression screening using alkaline phosphatase has been previously described for molecular characterization and cloning of receptors (see Flanagan and Cheng, Methods Enzymol 327:198-210 (2000)). A betatrophin-alkaline phosphatase (AP) fusion protein (using the gene ALPP) of mouse betatrophin and of human betatrophin were generated in HEK 293T cells. Betatrophin- AP fusion proteins (AP- hBT and AP-mBT) are secreted into the cell culture supernatant and can be applied to substrates of interest. [00149] For example, AP-BT-expressing supernatant can be incubated with cells transfected with a cDNA library followed by reaction with NBT-BCIP leading to a characteristic precipitation product, as shown in Figure 8A, to search for binding partners for betatrophin. Seven days after injection of the AP-hBT vector into mice led to the incorporation of AP-hBT into large protein complexes in native serum samples, as shown in the immunoblots using human betatrophin antibody (Phoenix anti-human Betatrophin purified IgG, G-051-55) in Figure 8B. In contrast, denatured serum samples from mice injected with AP-hBT showed a smaller protein. These data indicated that AP- hBT expressed by a plasmid in mice leads to incorporation of AP-hBT into large protein complexes, and thus in vitro screening with AP-hBT may allow identification of receptors for betatrophin.

[00150] A variety of cDNA libraries were screened with either AP-mBT or

AP-hBT, including a mouse pancreas library, a mouse liver library, and a human

ORFeome library (which expresses the complete set of open reading frames from the human genome). The mouse libraries were made using isolation of RNA from various tissues. The human ORFeome library was a gift from Dr. Doug Melton's lab.

[00151] Four binding partners were identified from cDNA library experiments. A class A macrophage scavenger receptor called Macrophage Receptor with Collagenous structure (MARCO) was identified from the mouse pancreas cDNA library. The reticulon 4 receptor (RTN4R), also known as Nogo-66 receptor (NgRl), was identified from the human ORFeome library. From the mouse liver cDNA library, Asgrl and hemopexin were identified as betatrophin binding proteins. Figure 8C shows confirmation data whereby GFP, MARCO, or RTN4R were expressed in Cos cells, cells were incubated with either AP-mBT or AP-hBT-containing supernatant, and then staining for alkaline phosphatase was done using NBT/BCIP (Sigma). The data in Figure 8C confirm that betatrophin conjugated to AP can bind both MARCO and RTN4R.

[00152] Knockout (KO) mice that lack MARCO were obtained from Lester

Kob2ik laboratory at Harvard School of Public Health. At five weeks of age, IPGTT experiments were performed as described in Example 1 to compare MARCO KO mice to wildtype mice. As shown in Figure 8D, MARCO KO mice had impaired IPGTT results as compared with wildtype. Data on the 3 mice from the phenotype database Mutant Mouse Resource and Research Centers (MMRRC; strain B6;129S5- Rtn4rtmlLex/Mmucd) indicates that RTN4R KO mice displayed an improved glucose tolerance at eight weeks as shown in Figure 8E, which is adapted from MMRRC. The in vitro data on MARCO and RTN4R on interactions with AP-hBT and in vivo data on alterations in IPGTT from KO animals support the hypothesis that MARCO and RTN4R may be functional binding partners for betatrophin that regulate glucose homeostasis.

[00153] The interaction of Asgrl and HPX were also evaluated. Cos cells expressing mouse Asgrl and hemopexin (HPX) were incubated with AP-mBT followed by reaction of AP with BCIP/NBT to produce a characteristic precipitate. Figures 9A and 9B show that no AP precipitation was seen when GFP-expressing Cos cells were incubated with AP-mBT or when mAsgrl -expressing Cos cells were incubated with AP alone. As shown in Figure 9C, incubation of mAsgrl -expressing Cos cells with AP-mBT followed by BCIP/NBT led to characteristic precipitate. Figure 9D shows that precipitate was also seen following incubation of mHPX-expressing Cos cells with AP- mBT followed by BCIP/NBT.

Example 4. Expression of Betatrophin in Subjects Exhibiting Hypoglycemia Following Gastric Bypass Surgery

[00154] Changes in betatrophin levels have been associated with conditions whereby beta cells proliferate. It is known that a percentage of subjects who undergo gastric bypass surgery later development hypoglycemia (see Rabiee 2011). Therefore, serum samples from subjects who developed hypoglycemia following gastric bypass were assessed for betatrophin levels.

[00155] Subjects who developed hypoglycemia following Roux-en-Y gastric bypass (RYGB) were assessed for islet size compared with healthy control subjects using previously described protocols (see Patti ME, et al., Diabetologia. 48(l l):2236-40 (2005)). As shown in Figure 10A, there was a dramatic beta cell expansion in hypoglycemic post- RYGB subjects compared with control subjects. Serum betatrophin levels were also measured from hypoglycemic post-RYGB subjects and controls using a betatrophin ELISA kit (Phoenix Pharmaceuticals, EK-051-60). Figures 10B and IOC present data from two independent experiments showing significantly higher levels of betatrophin in hypoglycemic post-RYGB subjects. Although levels of betatrophin in control subjects was approximately lng/ mL or less, levels as high as 50ng/ mL were seen in hypoglycemic post-RYGB subjects. These data suggest that betatrophin may be able to induce beta cell expansion over a relatively short period of time in post-gastric bypass subjects and that this beta cell expansion may be associated with the development of hypoglycemia. Thus, subjects hypoglycemic post gastric bypass surgery should be treated with betatrophin inhibitors to regulate blood glucose levels to within normal ranges.

Example 5. Characterization of short-term in vivo effects of MBP conjugated human betatrophin

[00156] MBP conjugated human betatrophin (MBP-hBT) was characteri2ed to better understand the effects of this agent on blood glucose.

[00157] Recombinant proteins were separated by SDS-PAGE, followed by

Commassie blue staining and Western blotting of MBP and MBP-hBT. Figure 11A shows staining of MBP and MBP-hBT with Commassie blue and immunoblotting (IB) with an anti-MBP antibody or an anti-liBT antibody. These results show that MBP-hBT was produced at the expected molecular weight. These MBP and MBP-hBT proteins were used for in vivo experiments to determine the physiologic effect of betatrophin.

[00158] Figure 11B shows an outline of the short-term in vivo experiments testing IV injection of MBP or MBP-hBT and their effects on blood glucose levels. CD1 mice were used at 10 weeks of age. Mice were dosed IV with 11 g of either MBP or MBP-hBT and blood samples were taken over 24 hours. At 2 and 3 hours after dosing, significant decreases in blood glucose were seen for the MBP-hBT-treated mice versus the MBP-treated mice (Figure 11C).

[00159] A dose-response experiment was performed to determine the blood glucose lowering effect induced by different doses of MBP or MBP-hBT. The change in basal blood glucose from baseline at 3 hours post-injection of recombinant protein was assessed. Figure 11D shows that doses as low as 5^g of MBP-hBT had a significant effect on lowering blood glucose.

[00160] Further experiments were conducted to better understand the time course of the effect of MBP-hBT. See, Figure HE. Mice were dosed with either MBP or MBP-hBT via cardiac puncture and sacrificed at 0, 1, 2, or 3 hours. A variety of parameters were measured including glucose (Figure 11F), insulin (Figure 11G), glucagon (Figure 11H), L-lactate (Figure 111), triglyceride (Figure 11J), and cholesterol (Figure UK, measured at 0 and 3 hours after injection). [00161] Significant decreases in glucose levels were seen after administration of MBP-hBT as compared to MBP at 2 and 3 hours post-injection (Figure 11F). These data confirm that MBP-hBT produces an acute lowering of glucose levels. Trends in other parameters, such as L-lactate, were seen that did not reach significance, which may be due to the relatively small sample sizes of the experiments.

[00162] Uptake of glucose by tissues following administration of

betatrophin was assessed using 2-deoxyglucose (2-DG), which is taken up by cells and then trapped intracellularly. To assess 2-DG uptake, non-fasted 10-week old CD1 male mice were administered 11 g MBP or MBP-hBT recombinant protein via tail vein injection, as shown in Figure 11L. Anesthesia was administered by intraperitoneal administration of pentobarbital at a dose of 100 mg/kg of body weight at 30 minutes after administration of MBP or MBP-hBT. At 60 minutes after administration of MBP or MBP-hBT, basal tail vein blood was sampled prior to 0.33 θ [³H] 2-deoxyglucose (2- DG) administration via the retro-orbital sinus. Tail vein blood samples were subsequently collected at 5, 10, 15, 25, 35, and 45 min after 2-DG injection for blood glucose level and [TTj concentration determination. Mice were sacrificed immediately by cervical dislocation after the last blood sampled time point, and tissues including subcutaneous (SQ) fat, visceral fat (VF), brown adipose tissue (BAT), liver, kidney, heart, intestine, brain, gastrocnemius, soleus, and triceps muscle were harvested and immediately fro2en in liquid nitrogen. Glucose uptake and utilization levels were determined by accumulation of [³FTJ2-deoxyglucose-6-P in tissues using a perchloric acid/BaOH-ZnS0₄ precipitation procedure.

[00163] 2-DG results (Figure 11M) show significantly greater glucose uptake in both BAT and heart following administration of MBP-hBT compared to MBP. Thus, betatrophin administration mediates rapid glucose uptake by BAT and heart.

[00164] Next, blood glucose levels of CD1 mice receiving MBP or MBP- hBT were tested via IPGTT as described in Figure 12A. CD1 mice were fasted for 5 hours, administered MBP or MBP-hBT, and then 1 hour later administered 2g/kg IP glucose. Figure 12B presents blood glucose levels over time. Administration of MBP- hBT resulted in significantly lower glucose levels at 30, 60, and 120 minutes after the glucose administration as compared to MBP. The area under the curve (AUC) over the course of the IPGTT experiment are shown in Figure 12C. These results further support the glucose-lowering effect of MBP-hBT. Significantly higher levels of insulin were also seen at 15 and 30 minutes after glucose challenge in the IPGTT test in mice that had been administered MBP-hBT versus MBP, as shown in Figure 12D.

[00165] Next, an oral glucose tolerance test (OGTT) was conducted in CD1 mice after administration of either MBP or MBP-hBT (Figure 12E). Pre-administration of MBP-hBT decreased blood glucose levels in the OGTT test (Figure 12F) and decreased the AUC (Figure 12G). Significantly higher levels of insulin were seen at 15 minutes after glucose challenge in mice after administration of MBP-hBT compared to mice receiving MBP (Figure 12H). These data support administration of betatrophin to quickly and efficiently mediate increased insulin levels and decreased blood glucose levels.

[00166] Effects of MBP-hBT on L-lactate were also measured from plasma collected by cardiac puncture at 30 minutes after glucose challenge in the OGTT, as described in Figure 121. Fasting mice were administered MBP or MBP-hBT, and oral gavage glucose was administered at 1 hour later. Plasma L-lactate levels were significantly higher in mice that had been administered MBP-hBT as compared to mice administered MBP (Figure 12J).

[00167] L-lactate is a byproduct of glucose uptake by liver. As a

consequence of glycolysis, L-lactate is released from liver into circulation. The increased plasma L-lactate at 30 minutes after administration of oral glucose in mice pre-treated with MBP-hBT suggests that betatrophin mediates rapid insulin-dependent uptake of glucose by liver.

[00168] Muscarinic acetylcholine receptors on beta cells regulate insulin release and thus mediate glucose homeostasis in vivo, see Gautam et al., CellMetab.

3(6):449-61(2006). Therefore, the role of muscarinic receptors in mediating the rapid effects of MBP-hBT was investigated.

[00169] CD1 mice were dosed with 11 μ_δ MBP or MBP-hBT, with some mice also being dosed with 1 mg/kg atropine, an inhibitor of muscarinic acetylcholine receptors. While administration of MBP-hBT led to a significant decrease in blood sugar at 2 and 3 hours after administration, this effect was blocked by administration of atropine at 10 min prior to MBP-hBT administration, as shown in Figure 13A. An IPGTT protocol as outlined in Figure 12A was used to assess the impact of atropine on a glucose challenge after MBP-hBT administration. Atropine was administrated 10 min prior to MBP-hBT injection and blocked the effect of MBP-hBT in an IPGTT model, as measured by glucose over time (Figure 13B) or the AUC (Figure 13C). Additionally, atropine blocked the effect of MBP-hBT to mediate an increase in blood insulin in the IPGTT model (Figure 13 D). Thus, these data are consistent with betatrophin mediating rapid insulin release from pancreatic beta cells through a muscarinic acetylcholine pathway, which can be blocked by treatment with atropine.

[00170] Next, the effects of betatrophin were assessed in the strepto2ocin

(STZ) diabetic mouse model. STZ is known to have preferential toxicity towards pancreatic beta cells and to induce diabetes. See, Figure 14A. CD1 mice were fasted for 4 hours and then intraperitoneally injected with 150mg/kg of STZ. At 2-3 days after STZ administration, blood glucose levels were tested in the STZ-treated mice. Those mice with blood glucose levels above 350mg/ dl were administered 11 g of MBP or MBP- hBT.]

[00171] Insulin levels in STZ-treated mice were lower (e.g., about 200ng/L), consistent with damage to pancreatic beta cells induced by STZ. Insulin levels were not different between diabetic mice administered MBP versus MBP-hBT at 24 hours after MBP or MBP-hBT administration (Figure 14B).

[00172] Even in the absence of a difference in insulin levels, treatment with

MBP-hBT led to lower glucose levels compared to treatment with MBP at 3 and 6 hours after recombinant protein administration in diabetic mice (Figure 14C). MBP-hBT administration led to a statistically significant change in basal blood glucose levels at 1, 2, 3, and 6 hours after administration (Figure 14 D). Thus, in a model of diabetes with damage to pancreatic beta cells and decreased insulin levels, betatrophin was still able to mediate a decrease in blood sugar levels that was significant within one hour of administration. These data indicate that betatrophin mediates a decrease in blood sugar levels via both insulin-dependent and insulin-independent mechanisms.

EQUIVALENTS

[00173] The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the embodiments. The foregoing description and Examples detail certain embodiments and describes the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the embodiment may be practiced in many ways and should be construed in accordance with the appended claims and any equivalents thereof.

[00174] As used herein, the term about refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term about generally refers to a range of numerical values (e.g., +/ -5-10% of the recited range) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). When terms such as at least and about precede a list of numerical values or ranges, the terms modify all of the values or ranges provided in the list. In some instances, the term about may include numerical values that are rounded to the nearest significant figure.

Claims

What is Claimed is:

1. A method of lowering blood glucose in a subject comprising administering betatrophin.

2. The method of claim 1, wherein blood glucose levels are lowered by about 72, 48, 24, 12, 6, 5, 4, 3, 2, or 1 hour(s) after administration.

3. The method of claim 1, wherein the subject has increased insulin levels.

4. The method of claim 1, wherein the subject does not have increased insulin levels.

5. The method of claim 1, wherein blood glucose levels are measured using a glucose tolerance test.

6. The method of claim 1, wherein after administration, glucose levels are increased in the liver, brown adipose tissue, or heart.

7. The method of claim 6, wherein glucose levels in the liver are measured by plasma L-lactate levels.

8. A method of increasing insulin levels in a subject comprising administering betatrophin.

9. The method of claim 8, wherein insulin levels in the blood are increased by about 72, 48, 24, 12, 6, 5, 4, 3, 2, or 1 hour(s) after administration.

10. A method of increasing the sensitivity of cells to insulin in a subject comprising administering betatrophin.

11. The method of claim 10, wherein insulin sensitivity of the cells of the body are increased by about 72, 48, 24, 12, 6, 5, 4, 3, 2, or 1 hour(s) after administration.

12. A method of decreasing the excretion of glucose from the liver in a subject comprising administering betatrophin.

13. The method of claim 12, wherein a decrease in the excretion of glucose from the liver into the blood is seen by about 72, 48, 24, 12, 6, 5, 4, 3, 2, or 1 hour(s) after administration.

14. A method of treating hyperglycemia in a subject comprising administering betatrophin.

15. The method of claim 14, wherein hyperglycemia is deemed treated when blood glucose levels are less than 200 mg/ dl within about 72, 48, 24, 12, 6, 5, 4, 3, 2, or 1 hour(s) after administration of betatrophin.

16. The method of any of claims 1-15, wherein the lowering of blood glucose, increasing insulin levels, increasing sensitivity of cells to insulin, and decreasing the excretion of glucose from the liver is independent of beta cell replication or proliferation.

17. The method of claim 14, wherein the treatment of hyperglycemia is independent of beta cell replication or proliferation.

18. The method of any of claims 1-17, wherein the subject has Type I diabetes mellitus, Type II diabetes, or metabolic syndromes.

19. The method of any of claims 1-18, wherein the subject has a blood sugar level higher than 11.1 mmol/liter or 200 mg/ dl.

20. The method of any of claims 1-19, wherein the subject is a mammal.

21. The method of claim 20, wherein the mammal is a human.

22. The method of any one of claims 1-21, wherein betatrophin is administered at a dose of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, 500, or 1000 /^k ·

23. The method of any of claims 1-22, wherein betatrophin is administered in combination with an additional treatment.

24. The method of claim 23, wherein the additional treatment is insulin.

25. The method of claim 24, wherein the insulin is a rapid-acting, intermediate-acting, or long-acting insulin.

26. The method of claim 23, wherein the additional treatment is a glucagon-like peptide analog or agonist, dipeptidyl peptidase-4 inhibitor, amylin analog, biguanide, thia2olidinedione, sulfonylurea, meglitinide, alpha-glucosidase inhibitor, or

sodium/ glucose transporter 2 inhibitor.

27. The method of any of claims 1-26, wherein a functional fragment of betatrophin is administered.

28. The method of claim 27, wherein the functional fragment of betatrophin comprises amino acids 22 to 76 of SEQ ID NO: 1, amino acids 48-76 of SEQ ID NO: 1, or amino acids 77 to 135 of SEQ ID NO: 1.

29. The method of any of claims 1-28, wherein betatrophin is in a complex with a lipoprotein.

30. The method of claim 29, wherein the lipoprotein is a high density lipoprotein (HDL) or a low density lipoprotein (LDL).

31. A method of decreasing blood glucose in a subject, comprising administering an agonist for a receptor that binds betatrophin.

32. The method of claim 31, wherein the receptor for betatrophin is MARCO, RTN4R, hemopexin, or Asgrl.

33. The method of claim 31, wherein the agonist is a small molecule, protein, or peptide.

34. A method of increasing blood glucose in a subject comprising administering a betatrophin inhibitor.

35. The method of claim 34, wherein the inhibitor blocks signaling of a betatrophin receptor.

36. The method of claim 35, wherein the betatrophin receptor is MARCO, RTN4R, hemopexin, or Asgrl.

37. The method of any of claims 34-36, wherein the inhibitor is an antibody, decoy receptor, small molecule, protein, or peptide.

38. The method of claim 37, wherein the betatrophin inhibitor is a muscarinic receptor antagonist.

39. The method of claim 38, wherein the muscarinic receptor antagonist is atropine.

40. The method of any of claims 34-39, wherein the inhibitor decreases beta cell proliferation or replication.

41. The method of any of claims 34-40, wherein the subject has hypoglycemia following bariatric surgery.

42. The method of claim 41, wherein the bariatric surgery is gastric banding.

43. The method of claim 41, wherein the bariatric surgery is a gastric bypass surgery.

44. The method of claim 41, wherein the gastric bypass surgery is Roux-en-Y gastric bypass.

45. A method of lowering blood glucose in a subject comprising administering betatrophin and HDL.

46. A method of increasing insulin levels in a subject comprising administering betatrophin and HDL.

47. A method of increasing the sensitivity of cells to insulin in a subject comprising administering betatrophin and HDL.

48. A method of decreasing the excretion of glucose from the liver in a subject comprising administering betatrophin and HDL.

49. A method of treating hyperglycemia in a subject comprising administering betatrophin and HDL, wherein hyperglycemia is treated when blood glucose levels are less than 200 mg/ dl.

50. The method of any of claims 40-44, wherein the lowering of blood glucose, increasing insulin levels, increasing sensitivity of cells to insulin, and decreasing the excretion of glucose from the liver is independent of beta cell proliferation or replication.

51. The method of claim 45, wherein the lowering of blood glucose, increasing insulin levels, increasing sensitivity of cells to insulin, and decreasing the excretion of glucose from the liver is seen within about 72, 48, 24, 12, 6, 5, 4, 3, 2, or 1 hour(s) after administration of betatrophin.

52. The method of any of claims 45-51, wherein the lowering of blood glucose, increasing insulin levels, increasing sensitivity of cells to insulin, and decreasing the excretion of glucose from the liver is dependent of beta cell proliferation or replication.

53. The method of claim 52, wherein the lowering of blood glucose, increasing insulin levels, increasing sensitivity of cells to insulin, and decreasing the excretion of glucose from the liver is seen only after at least 72 hours.

54. Use of betatropin for the preparation of a medicament to lower blood glucose, increase insulin levels, increase sensitivity of cells to insulin, and decrease the excretion of glucose from the liver.