WO2016138132A1 - Dipeptidyl peptidase-iv(dpp4) inhibitors, methods and compositions for suppressing adipose tissue inflammation - Google Patents

Abstract

The invention relates generally to blocking dipeptidyl-peptidase 4 (DPP4) secretion/synthesis in hepatic cells, which in turn inhibits or blocks adipose tissue inflammation. This process is involved in metabolism and type 2 diabetes, and will be useful for developing new DPP4 inhibitors and for treating or preventing conditions such as type 2 diabetes, metabolic syndrome, obesity, insulin resistance, as well as pre diabetes, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, diabetic dyslipidemia, fatty liver disease including non-alcoholic fatly liver disease (NASH), and atherosclerosis.

Description

DIPEPTIDYL PEPTIDASE-IV (DPP4) INHIBITORS, METHODS AND COMPOSITIONS FOR SUPPRESSING ADIPOSE TISSUE INFLAMMATION CROSS-REFERENCE TO RELATED APPLICATIONS

This present application claims priority to U.S. Provisional Patent Application Ser. No. 62/120,549 filed February 25, 2015, which is incorporated herein by reference in its entirety. STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant number POl HL087123 awarded by the National Institutes of Health. The government may have certain rights in this invention.

FIELD OF THE INVENTION

The invention relates generally to blocking dipeptidyl-peptidase 4 (DPP4) secretion/synthesis in hepatic cells, which in turn inhibits or blocks adipose tissue inflammation. This process is involved in metabolism and type 2 diabetes, and will be useful for developing new DPP4 inhibitors and for treating or preventing conditions such as type 2 diabetes, metabolic syndrome, obesity, insulin resistance, as well as pre diabetes, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, diabetic dyslipidemia, fatty liver disease including non-alcoholic fatty liver disease (NASH), and atherosclerosis.

BACKGROUND OF THE INVENTION

Dipeptidyl peptidase-IV (DPP-IV, DPP-4, or DPP4) is a serine protease that belongs to a group of post-proline/aianine cleaving amino-dipeptidases. DPP4 catalyzes the release of an N- terminal dipeptide only from proteins with N-terminal penultimate proline or alanine.

The physiological role of DPP4 has not been established fully. It is believed to play an important role in neuropeptide metabolism, T-cell activation, gastric ulceration, functional dyspepsia, obesity, appetite regulation, impaired fasting glucose (IFG), and diabetes. In particular, DPP4 has been implicated in the control of glucose metabolism because its substrates include the insulinotropic hormones, glucagon like peptide-1 (GLP-1) and gastric inhibitory peptide (G1P), which are inactivated by removal of their two N-terminal amino acids.

In vivo administration of synthetic inhibitors of DPP4 prevents N-terminal degradation of GLP-1 and GIP, resulting in higher plasma concentrations of tliese hormones, increased insulin secretion and, therefore, improved glucose tolerance. Therefore, such inhibitors are being used for the treatment of patients with type II diabetes, a disease characterized by decreased glucose tolerance and insulin resistance.

Diabetic dyslipidemia is characterized by multiple lipoprotein defects, including moderately high serum levels of cholesterol and triglycerides, small LDL particles, and low levels of HDL cholesterol. The results of recent clinical trials reveal beneficial effects of cholesterol- lowering therapy in diabetic and nondiabetic patients, thus supporting increased emphasis on treatment of diabetic dyslipidemia. This need for intensive treatment of diabetic dyslipidemia was advocated by the National Cholesterol Education Program's Adult Treatment Panel III.

Obesity is a well-known risk factor for the development of many very common diseases such as atherosclerosis, hypertension and diabetes. The incidence of obese people and thereby also these diseases is increasing throughout the entire industrialized world. Except for exercise, diet and food restriction, no convincing pharmacological treatment for reducing body weight effectively and acceptably currently exist. However, due to its indirect but important effect as a risk factor in mortal and common diseases it will be important to find treatment for obesity or appetite regulation. Even mild obesity increases the risk for premature death, diabetes, hypertension, atherosclerosis, gallbladder disease and certain types of cancer. In the industrialized western world the prevalence of obesity has increased significantly in the past few decades. Because of the high prevalence of obesity and its health consequences, its prevention and treatment should be a high public health priority.

At present a variety of techniques are available to effect initial weight loss. Unfortunately, initial weight loss is not an optimal therapeutic goal. Rather, the problem is that most obese patients eventually regain their weight. An effective means to establish and/or sustain weight loss is the major challenge in the treatment of obesity today.

Diabetes mellitus is a metabolic disorder characterized by recurrent or persistent hyperglycemia (high blood glucose) and other signs, as distinct from a single disease or condition. Glucose level abnormalities can result in serious long-term complications, which include cardiovascular disease, chronic renal failure, retinal damage, nerve damage (of several lands), microvascular damage and obesity.

Type 1 diabetes, also known as Insulin Dependent Diabetes Mellitus (IDDM), is characterized by loss of the insulin-producing ,beta.-cells of the islets of Langerhans of the pancreas leading to a deficiency of insulin. Type-2 diabetes previously known as adult-onset diabetes, maturity-onset diabetes, or Non-Insulin Dependent Diabetes Mellitus (NIDDM)— is due to a combination of increased hepatic glucose output, defective insulin secretion, and insulin resistance or reduced insulin sensitivity (defective responsiveness of tissues to insulin).

Chronic elevation of blood glucose level leads to damage of blood vessels. In diabetes, the resultant problems are grouped under "microvascular disease" (due to damage of small blood vessels) and "macrovascular disease" (due to damage of the arteries). Examples of microvascular disease include diabetic retinopathy, neuropathy and nephropathy, while examples of macrovascular disease include coronary artery disease, stroke, peripheral vascular disease, and diabetic myonecrosis.

Diabetic retinopathy, characterized by the growth of weakened blood vessels in the retina as well as macular edema (swelling of the macula), can lead to severe vision loss or blindness. Retinal damage (from microangiopathy) makes it the most common cause, of blindness among non-elderly adults in the US. Diabetic neuropathy is characterized by compromised nerve function in the lower extremities. When combined with damaged blood vessels, diabetic neuropathy can lead to diabetic foot. Other forms of diabetic neuropathy may present as mononeuritis or autonomic neuropathy. Diabetic nephropathy is characterized by damage to the kidney, which can lead to chronic renal failure, eventually requiring dialysis. Diabetes mellitus is the most common cause of adult kidney failure worldwide. A high glycemic diet (i.e., a diet that consists of meals that give high postprandial blood sugar) is known to be one of the causative factors contributing to the development of obesity.

DPP4 inhibitors (DPP4-I) are widely used in humans with type 2 diabetes (T2D), with the major mechanism of action presumed to be the stabilization of GLP1, which promotes insulin secretion from pancreatic beta cells. However, DPP4-I have other beneficial effects on metabolism and the mechanisms are not known.¹ Moreover, circulating levels of DPP4 correlate with BMI and insulin resistance in humans." In this context, one of the key hallmarks of T2D is visceral adipose tissue (VAT) inflammation.³ Studies have shown that VAT inflammation is an important exacerbating feature of insulin resistance and T2D and that blocking AT inflammation can improve metabolism.⁴ Three studies, including 2 using des-fluoro-sitagliptin, have shown that treating diabetic mice with DPP4-I decreased adipose inflammation.^5"' However, whether this effect is due to inhibition of an inflammatory effect of DPP4 on adipose tissue and, if so, the tissue source and regulation of DPP4 for this effect and the target cell and mechanisms of how DPP4 might cause adipose inflammation are not known.

Several compounds have been shown to inhibit DPP4, but all of these have limitations in relation to the potency, stability, selectivity, toxicity, and/or pharmacodynamic properties. Such compounds have been disclosed, for example, in U. S. Patent Nos.: 6,699,871; 7,125,873; and 7,326,708 (Merck) WO 98/19998, WO 00/34241, U.S. Pat. No. 6,124,305 (Novartis AG), U.S. Pat. No. 6,303,661 (Probiodrug) and WO 99/38501 (Trustees of Tufts University).

Thus, there is an ongoing need for identifying new DPP4-I, as svell as for understanding the mechanism of action of DPP4-I in order to identify new indications for treatment, and for identifying possible drug combinations and methods for treating conditions in which visceral adipose tissue (VAT) inflammation plays a role, including type II diabetes, prediabetes, obesity, metabolic disease, insulin resistance, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, diabetic dyslipidemia, and potentially other metabolic diseases such as fatty liver disease, including non-alcoholic fatty liver disease (NASH), and atherosclerosis.

SUMMARY OF THE INVENTION

In certain embodiments, the present invention relates to a method for decreasing adipose tissue inflammation, comprising blocking DPP4 secretion from hepatocytes in the tissue of a subject in need thereof.

In certain embodiments, the blocking comprises administering an antagonist of DPP4.

In certain embodiments, the blocking comprises hepatic cell-specific genetic knockdown or knockout of DPP4.

In certain embodiments, the method further comprises inhibiting PAR2 binding to DPP4 in the tissue. In certain embodiments, inhibiting PAR2 binding comprises contacting the tissue with an antagonist of PAR2.

In certain embodiments, the present invention relates to a method for decreasing adipose tissue inflammation comprising administering to a subject in need thereof, a compound that blocks DPP4 secretion from hepatocytes. In certain embodiments, the present invention relates to a method for preventing or alleviating obesity comprising administering to a subject in need thereof, a compound that blocks DPP4 secretion from hepatocytes.

In certain embodiments, the present invention relates to a method for preventing or alleviating metabolic syndrome comprising administering to a subject in need thereof, a compound that blocks DPP4 secretion from hepatocytes. In certain embodiments, the subject is suffering from one or more conditions selected from the group consisting of: type 2 diabetes, metabolic syndrome, pre diabetes, obesity, insulin resistance, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, diabetic dyslipidemia, fatty liver disease including non-alcoholic fatty liver disease (NASH), and atherosclerosis.

In certain embodiments, the compound or blocking is prophylactically administered prior to symptoms normally associated with conditions selected from the group consisting of type 2 diabetes, metabolic syndrome, pre diabetes, obesity, insulin resistance, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, diabetic dyslipidemia, fatty liver disease including non-alcoholic fatty liver disease (NASH), and atherosclerosis.

In additional embodiments, the method further improves insulin sensitivity. In additional embodiments, the method further suppresses VAT inflammation.

In certain embodiments, the present invention relates to a method for identifying a compound that blocks DPP4 secretion from hepatocytes comprising: a) incubating a hepatocyte culture with palmitate to induce DPP4 secretion, b) measuring secreted DPP4 in the induced hepatocyte culture, c) contacting and incubating the induced hepatocytes with a test compound, and d) measuring secreted DPP4 in the induced hepatocyte culture following the contacting and incubating step c), wherein a decrease in secreted DPP4 in the induced hepatocyte culture indicates a compound that blocks DPP4 secretion from induced hepatocytes, and wherein the decrease is a level similar to a reference basal DPP4 level observed in medium from non- induced hepatocytes. In certain embodiments the measuring is by ELISA.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-F illustrate that hepatocyte (HC)-specific CaMKII deletion lowers visceral adipose tissue (VAT) inflammation in diet-induced obese (DIO) mice, whereas hepatic and systemic inflammation are not affected. Thirteen-week-old DIO Camk2g^m mice were treated with adeno-associated viruses (AAV) containing either hepatocyte-specific TBG-Cre recombinase or the control vector (TBG-LacZ) to obtain HC-CaMKII KO and WT mice, respectively. Fig. 1A shows representative images of VAT sections stained by immunohistochemistry with an antibody against the macrophage marker F4/80 after three weeks of AAV treatment. Figs. lB-C are graphs showing shVAT and liver F4/80, Mcpl, and Tnf-a mRNA levels were assayed by RT-qPCR after three weeks of AAV treatment. Fig. ID are graphs showing plasma 1L-6 and Tnf-a levels determined using a luminex assay system after three weeks of AAV treatment. Fig. IE are graphs showing total blood monocyte counts that were monitored from DIO WT or HC-CaMKII KO mice (n = 3) after three weeks of AAV treatment. The 1HC data shown in Fig. IF used the Thirteen-week-old DIO Camklg^ mice treated as in Fig. 1A. Three weeks later, Ly6c^h' monocytes were labeled with fluorescent beads in vivo in WT and HC-CaMKII KO DIO mice, and then bead-labelled ATMs were counted in VAT sections (n = 4, *, p < 0.05; mean ± SEM.).

Figures 2A-D show that the suppression of VAT inflammation in DIO HC-

CaMKII KO mice is independent of the improvement in systemic metabolism. Fig. 2A-B are graphs showing rsults from twelve-week-old DIO Camk2g^m mice treated with TBG-Cre or TBG-LacZ to obtain HC-CaMKII KO and WT mice, respectively. Five days later half of the TBG-Cre mice received adeno-TRB3 to restore the metabolic defect, while the other half received adeno-LacZ control. 5 hr fasting and fasted-refed blood glucose and plasma insulin were assayed after four weeks of AAV treatment. Fig. 2C shows representative images of hematoxyiin-eosin (H&E) staining of VAT sections after four weeks of AAV treatment. Fig. 2D is a graph showing results of VAT F480, Mcpl, and Tnf-a mRNA levels assayed by RT- qPCR after four weeks of adenovirus treatment (n = 4; *, p < 0.05; mean + SEM). Figures 3A-C show that suppression of VAT inflammation by HC CaMKII deficiency is abrogated by restoring ATF4, whereas liver inflammation is not affected. Fig. 3A shows images of VAT sections from twelve-week-old Camk2g ^x DIO mice treated with TBG-Cre or TBG-LacZ to obtain HC-CaMKII KO and WT mice, respectively. Two days later half of the TBG-Cre treated mice received adeno-ATF4, while the other half received adeno-LacZ control. Representative images of VAT sections stained by immunohistochemistry with an antibody against the macrophage marker F4/80 after four weeks of adenovirus treatment are shown. Figs. 3B-C are graphs showing results from VAT and liver F480, Mcpl, and Tnf-a mRNA levels assayed by RT-qPCR after four weeks of adenovirus treatment, (n = 4: bars with different symbols are different from each other and from control, p < 0.05; mean + SEM).

Figures 4A-C show that ATF4 expression in livers of lean mice is sufficient to induce VAT Mcpl. Figs. 4A-B are western blots from 10- week-old WT lean mice treated with adeno-LacZ (Ad-LacZ) or adeno-ATF4 (Ad-ATF4) for two weeks and then liver and VAT extracts were assayed for ATF4 and β-actin by immunoblot. Fig. 4C is a graph showing VAT Mcpl mRNA levels assayed by RT-qPCR after two weeks of adenovirus treatment (n = 3 or 4; *, p < 0.05; mean + SEM).

Figures 5A-G show that HC-derived VAT inflammatory activity can be detected in plasma using an ex-vivo adipose stromai-vascular-fraction (SVF) assay. Fig. 5A are representative images of VAT sections from the VAT of lean and obese mice that were stained with H & E are shown. Fig. 5B is a graph showing VAT Mcpl mRNA levels assayed by RT-qPCR. Fig. 5C is a graph showing VAT stromal vascular fraction (SVF) cells obtained from a DIO mouse incubated with a medium containing 10% of lean or DIO mice plasma for 18 h and then Mcpl mRNA levels were assayed by RT-qPCR. Fig. 5D is a graph showing data from thirteen- week-old DIO Camk2g^ft' mice were treated with adeno- associated viruses (AAV) containing either hepalocyte-specific TBG-Cre recombinase or the control vector (TBG-LacZ) to obtain HC-CaMKII KO and WT mice, respectively. Mcpl mRNA levels in VAT of WT or HC-CaMKII KO DIO mice were assayed by RT-qPCR after three weeks of AAV treatment (n = 4). Fig. 5E is a graph showing data from thirteen-week- old DIO Camklg^ mice were treated as in Fig. 5D for three weeks. VAT SVF cells from a DIO mouse were incubated with a medium containing 10% of plasma obtained from WT or HC-CaMKII KO DIO mice for 18 h and then Mcpl mRNA levels were assayed by RT- qPCR. Fig. 5F is a graph showing data from twelve-week-old Camk2g^m DIO mice treated with TBG-Cre or TBG-LacZ to obtain HC-CaMKII KO and W^rT mice, respectively. Two days later half of the TBG-Cre treated mice received adeno-ATF4, while the other half received adeno-LacZ control for four weeks. VAT SVF cells from a DIO mouse were incubated with medium containing 10% of plasma obtained from indicated groups of mice and Mcpl levels were assayed by RT-qPCR. Fig. 5G is a graph showing data from 10-week- old WT lean mice were treated with adeno-LacZ (Ad-LacZ) or adeno-ATF4 (Ad-.ATF4) for three weeks. VAT SVF cells from a DIO mouse were incubated with medium containing 10% of pooled plasma obtained from indicated groups of mice and Mcpl levels were assayed by RT-qPCR (bars with different symbols are different from each other and from control, p < 0.05; mean ± SEM). Figures 6A-J show that plasma dipeptidyl-peptidase 4 (DPP-4) mediates VAT inflammation in obesity. Fig. 6A is a graph showing data from VAT SVF cells from a DIO mouse incubated with a medium containing 10 % control or heat-inactivated (HI) plasma from the indicated groups of mice for 18 h and then Mcpl mRNA levels were assayed by RT- qPCR. Fig. 6B is a graph of UV protein chromatogram obtained after fractionation of lean and DIO mice plasma using size exclusion gel filtration fast flow chromatography is shown. Fig. 6C is a graph showing data from VAT SVF cells obtained from a DIO mouse were incubated with a medium containing 10 % of unfractionated plasma or plasma FPLC fractions corresponding to chromatogram peak in the 150 kDa-400 kDa range of protein separation from lean or DIO mice for 18 h and then Mcpl rnRNA levels were assayed by RT- qPCR. Fig. 6D-F are graphs showing data from tsvelve-week-old Camk2g^m DIO mice were treated with TBG-Cre or TBG-LacZ to obtain HC-CaMKII KO and WT mice, respectively. Two days later half of the TBG-Cre treated mice received adeno-ATF4, while the other half received adeno-LacZ control for four weeks. VAT SVF cells obtained from a DIO mouse were incubated with medium containing 10% of unfractionated plasma or plasma FPLC fractions corresponding to chromatogram peak in the 150 kDa-400 kDa range of protein separation from WT, HC-CaMKll KO or ATF4 restored HC-CaMKII KO DIO mice for 18 h and then Mcpl mRNA levels were assayed by RT-qPCR. Fig. 6G is a blot from obese DIO mouse plasma Mcpl inactive FPLC fractions (32, 34, 40, 41, 42, 46, 47, 48, 49, 58 or 61) or Mcpl active FPLC fractions (43, 44 or 45) were run on SDS PAGE reducing gel, stained with coomassie staining solution and destained using HPLC grade water. Two of Mcpl inactive fraction proteins (40 and 47) and one Mcpl active fraction proteins (44) were divided into 3 parts as marked and then subjected to in-gel trypsin digestion overnight. Tryptic-digested peptides were subjected to Fusion Tribrid Mass Spectrometer+Easy-nLC 1000 for global proteome analysis. Fig. 6H is a graph showing DPP-4 levels detected after spectral analysis of indicated DIO mouse plasma fractions using LC-MS/MS. Fig. 61 is a graph showing DPP- 4 activity measured in lean and DIO mice plasma using a f!uorometric DPP-4 activity assay. Fig. 6J is a graph showing data from VAT SVF cells obtained from a DIO mouse incubated for 18 h with a medium containing 10% of DIO mice plasma with indicated doses of DPP-4 inhibitor (KR62436) after 1 hour pretreatment and Mcpl levels were assayed (bars with different symbols are different from each other and from control, p < 0.05; mean ± SEM).

Figures 7A-E are graphs showing that the CaMKII-ATF4 pathway regulates hepatic but not VAT DPP-4 expression and activity. Fig. 7A-B are graphs showing results from twelve- week-old Camklg^ DIO mice treated with TBG-Cre or TBG-LacZ to obtain HC-CaMKII KO and WT mice, respectively. Two days later half of the TBG-Cre treated mice received adeno-ATF4, while the other half received adeno-LacZ. Four weeks later, liver and VAT Dpp-4 mRNA levels were assayed by RT-qPCR. Fig. 7C is a graph showing DPP- 4 activity measured in liver lysates obtained from VVT, HC-CaMKII KO or ATF4 restored HC-CaMKII KO DIO mice using a fluorometric DPP-4 activity assay. Fig. 7D graph is similar to Fig. 7C except that VAT lysate was used. Fig. 7E is a graph showing data from primary hepalocvtes from WT mice transduced with adeno-LacZ (Ad-LacZ) or adeno-ATF4 at an moi of 10 and 24 h later, Dpp4 mRNA levels were assayed by RT-qPCR (bars with different symbols are different from each other and from control, p < 0.05; mean ± SEM).

Figures 8A-E show that hepatic CaMKII-ATF4 pathway regulates plasma DPP- 4 activity. Fig. 8A graph shows results from twelve-week-old Camk2gfl/fl DIO mice treated with TBG-Cre or TBG-LacZ to obtain HC-CaMKII KO and WT mice, respectively. Two days later half of the TBG-Cre treated mice received adeno-ATF4, while the other half received adeno-LacZ. Four weeks later, plasma from WT, CaMKII KO or ATF4 restored HC-CaMKII KO DIO mice were used to assay activity (Fig. 8A) of DPP-4 using a fluorometric DPP-4 activity assay . Figs. 8B-D are graphs showing results from twelve-week- old Camk2gfl/fl DIO mice treated as in (Fig. 8A) for four weeks. DPP-4 activity was measured in plasma FPLC fractions corresponding to chromatogram peak in the 150 kDa-200 kDa range of protein separation from WT, HC-CaMKII KO or ATF4 restored HC-CaMKll KO DIO mice using a fluorometric DPP-4 activity assay. Fig. 8E is a graph showing results from 10-week-old WT lean mice treated with adeno-LacZ (Ad-LacZ) or adeno-ATF4 (Ad- ATF4). Two weeks later, plasma was used to monitor DPP-4 activity (Fig. 8E) using a fluorometric DPP-4 activity assay (bars with different symbols are different from each other and from control, p < 0.05; mean + SEM).

Figures 9A-F show that obese plasma triggers inflammation in M<])s and is dependent on DPP4 and inflammatory signaling intermediates. Fig. 9A is a graph showing results from obese VAT SVF cells, adipose tissue macrophages (ATMs), non-ATMs or mixture of ATMs and non-ATMs which were incubated for 18 h with a medium containing 10 % of either lean or obese mice plasma and then Mcpl mRNA levels were assayed by RT-qPCR (n = 3). Figs. 9B-D are graphs showing results from obese VAT SVF cells, primary peritoneal macrophages or bone marrow derived macrophages (BMDM) which were incubated for 18 h with a medium containing 10% of lean or obese mice plasma and Mcpl levels were assayed by RT-qPCR. (n= 3). Fig. 9E is a graph showing results from BMDM treated with 10 μΜ of DPP-4 inhibitor (KR62436) 1 hour prior to addition of 10% lean or obese mice plasma and then Mcpl mRNA levels were assayed by RT-qPCR. (n = 3). Fig. 9F is a graph showing results from SVF cells from DIO mouse VAT were pretreated with indicated MAPK inhibitors (10 μΜ), PI3K inhibitor (10 μΜ, PKA inhibitor (5 μΜ) or NFKB inhibitor ( 10 μΜ) for 1 h before incubation with 10 % DIO mouse plasma for 18 h. Mcpl mRNA levels were then assayed by RT-qPCR (n = 3; bars with different symbols are different from each other and from control, p < 0.05; mean ± SEM). Figures 10A-D show that plasma from obese humans promotes VAT inflammation. Fig. 10A is a graph showing data from obese mouse VAT SVF cells incubated for 18 h with 10% plasma obtained from lean or obese individuals and then Mcpl mRNA levels were assayed by RT-qPCR. Fig. 10B is a graph showing data from obese mouse VAT SVF cells incubated with control plasma or heat inactivated (HI) plasma from lean or obese individuals for 18 h and then Mcpl mRNA levels were assayed by RT-qPCR. Fig. IOC is a graph showing data from UV protein chromatogram obtained after fractionation of lean and obese human serum samples using size exclusion gel filtration fast flow chromatography. Fig. 10D is a graph showing data from obese mouse VAT SVF cells incubated with unfractionated serum or FPLC fractions corresponding to chromatogram peak in the 150 kDa-400 kDa range of protein separation from lean or obese human serum and Mcpl mRNA levels were assayed by RT-qPCR (bars with different symbols are different from each other and from control, p < 0.05; mean ± SEM).

Figures 11A-B illustrate that HC specific deletion of ATF4 suppresses VAT inflammation. Seventeen week old Atf4^{fl il} mice were treated with adeno-associated virus (AAV) containing either hepatocyte-specific TBG-Cre recombinase or the control vector (TBG-LacZ) to obtain HC-ATF4 KO and WT mice. Fig. 11A shows representative images of VAT sections stained by immunohistochemistry with an antibody against the macrophage marker F4/80 after three weeks of AAV treatment. Fig. 11B is a graph showing quantification of crown like structure (CLS) macrophages, (n = 12 for WT and n = 1 1 for HC-ATF4 KO, *, p < 0.05; mean ± SEM).

Figures 12A-G show that hepatocyte specific deletion of DPP4 improves insulin sensitivity in DIO mice. Seventeen week old DIO mice were injected with adeno-associated virus (AAV) containing either ShDPP4 or the control vector (ShLacZ). Four weeks later liver extracts (Fig. 12A) or VAT extracts (Fig.l2B) were assayed for DPP-4 protein levels by immunoblotting or Dpp-4 mRNA levels by RT-qPCR (n = 5; *, p < 0.05; mean ± SEM). Fig. 12C graph shows plasma DPP-4 activity levels in control vs AAV8-ShDPP4 treated DIO mice using a f!uorometric DPP-4 activity assay, (n = 5; *, p < 0.05; mean ± SEM). Fig. 12D is a graph showing 5h fasting plasma glucose levels in control and AAV8-ShDPP4 treated DIO mice. Fig 12E shows the results obtained from glucose tolerance test (GTT) performed on control and AAV8-ShDPP4 treated DIO mice. Mice were fasted overnight and then injected with lg D-glucose/kg of mice and plasma glucose was measured at regular lime intervals as shown. Fig. 12F is a graph showing plasma insulin levels obtained after 5h fasting of control or AAV8-ShDPP4 treated DIO mice. Fig. 12G is a graph showing insulin tolerance test (ITT) in control and AAV8-ShDPP4 treated DIO mice. Mice were fasted for 5h before injecting 0.7 IU/kg of insulin per mouse and plasma glucose levels were monitored over indicated time intervals, (n = 5; *, p < 0.05; mean ± SEM). Figures 13A-D show that liver specific deletion of DPP-4 lowers VAT inflammation. Seventeen week old DIO mice were injected with adeno-associated vims (AAV) containing either ShDPP4 or the control vector (ShLacZ). Fig. 13A shows representative images of VAT sections stained by immunohistochemistry with an antibody against the macrophage marker F4/80 after four weeks of AAV8-LacZ or AAV8-ShDPP4 treatment. Fig. 13B is a graph showing results obtained from quantification of crown like structure (CLS) macrophages in VAT sections. Fig. 13C is a graph showing results from VAT ¥4/80, Mcpl, Tnfa and lUh mRNA levels assayed by RT-qPCR after four weeks of AAV8-LacZ or AAV8-ShDPP4 treatment, (n = 5; bars with different symbols are different from each other and from control, p < 0.05; mean ± SEM). Fig. 13D shows an immunoblol obtained from VAT tissues of either control or AAV8-ShDPP4 treated obese mice. VAT tissue extracts were obtained after four weeks of adeno associated virus treatment and activation of Akt pathway was assayed after 4 min of portal vein injection of 0.7 IU/kg of insulin.

Figures 14A-C show that obese pIasma-DPP-4 triggered inflammation in stromal vascular fraction (SVF) cells and M(j>s is reduced by PAR2 inhibition. Fig. 14A is a graph showing results from obese VAT SVF cells, which were incubated with 20 μΜ of PAR2 antagonist peptide (FSLLRY-NH2) lh prior to addition of 10% of lean or obese mice plasma and then Mcpl mRNA levels were assayed bt RT-qPCR. (n = 5). Fig. 14B is a graph showing results from bone marrow derived macrophage (BMDM), which were treated with either PAR2 inhibitor BB83 (10 μΜ) or with PAR2 antagonist peptide (20μΜ) for lh before incubation with obese plasma and then Mcpl mRNA levels were assayed using RT-qPCR. (n = 4). Fig 14C shows data obtained from obese VAT SVF cells. Obese plasma was incubated with either protein G beads conjugated anti-DPP4 antibody or control IgG for 2h on rotating shaker. Immunoprecipitated DPP-4 was then eluted using elution buffer. Obese VAT SVF cells were incubated with PAR2 antagonist peptide (20μΜ) for lh before incubation with IP- control or 1P-DPP4 proteins for 4h and then Mcpl mRNA levels were assayed using RT- qPCR. (n = 3; bars with different symbols are different from each other and from control, p < 0.05; mean ± SEM).

Figures 15A-C show that DPP4 needs to be complexed with one or more plasma proteins to cause VAT inflammation. Fig. ISA is a graph showing data from obese VAT SVF cells, which were either treated with mouse rDPP-4 alone or in combination with obese mouse plasma for 4h and then Mcpl mRNA levels were assayed using RT-qPCR. Fig. 15B shows results obtained from obese VAT SVF cells which were treated with either obese plasma, DPP-4 depleted obese plasma or combination of DPP-4 depleted obese plasma and mouse rDPP-4 or mouse rDPP4 alone for 4h and then Mcpl mRNA levels were assayed using RT-qPCR. Fig.l5C is a graph showing data obtained from obese VAT SVF cells, which were incubated with either IP-Control or IP-DPP-4 for 4h and then assayed for Mcpl mRNA levels using RT-qPCR. (n = 3; bars with different symbols are different from each other and from control, p < 0.05; mean ± SEM).

Figures 16A-F show that Dpp4 mRNA could be spliced and is secreted out of HCs. Fig. 16A graphically represents Dpp4 mRNA and summarizes strategy to design primers for determining spliced forms of Dpp4 mRNA. Fig 16B is graph showing in vivo expression of Dpp4 mRNA in liver samples obtained from either WT or HC-CaMKII KO or ATF4 restored DIO obese mice. Dpp4 mRNA levels were assayed using either N-lerminal primers or C terminal primers by RT-qPCR. (n = 4). Fig. 16C shows results obtained from mouse primary HCs, which were treated with 300μΜ of palmitate for indicated time points and medium DPP4 activity levels was assayed using a fluorometric DPP-4 activity assay (n = 6). Fig. 16D is a graph obtained from FACS analysis of surface DPP4 on mouse primary HCs, which were treated as mentioned in panel Fig 16C. (n= 6). Fig. 16E shows results obtained from mouse primary HCs, which were treated with Monensin (2 μΜ) or Tapi2 (5 μΜ) lh prior to palmitate incubation for 20h. Secreted DPP4 in medium was assayed using a fluorometric DPP-4 activity assay (n = 6). Fig. 16F is a graph showing results obtained from mouse primary HCs, which were treated as mentioned in panel Fig 16E. FACS analysis was performed to assay surface levels of DPP4 on mouse primary HCs. (n = 6; *, p < 0.05; mean + SEM). DETAILED DESCRIPTION

In certain embodiments, the present invention relates to methods for decreasing adipose tissue inflammation comprising administering to a subject, a compound that blocks DPP4 secretion from hepatocytes. Blocking DPP4 synthesis in hepatocytes, has the downstream effect of blocking adipose tissue inflammation, which is beneficial in many aspects of metabolism including type 2 diabetes, as well as metabolic syndrome and conditions in which visceral adipose tissue (VAT) inflammation plays a role, including, pre diabetes, obesity, insulin resistance, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, diabetic dysMpidemia, and potentially other metabolic diseases such as fatty liver disease including non-alcoholic fatty liver disease (NASH), and atherosclerosis.

In certain embodiments, the present invention relates to a method for preventing or alleviating obesity comprising administering to a subject, a compound that blocks DPP4 secretion from hepatocytes. In certain embodiments, the present invention relates to a method for preventing or alleviating metabolic syndrome comprising administering to a subject, a compound that blocks DPP4 secretion from hepatocytes. In certain cases, the method further improves insulin sensitivity. In certain cases, the method further suppresses VAT inflammation.

The present data show that liver DDP4 synthesis and circulating levels of DDP4 are suppressed in obese mice when hepatocyte ATF4 is silenced. This data is useful for elucidating the mechanisms of ATF4-mediated induction of DPP4.

The present data also show that DPP4 activates inflammatory pathways in macrophages and provides further elucidation of the mechanism involved in this process.

The present data also shows that blocking DPP4 secretion from hepatocytes in obese mice decreases adipose tissue inflammation and improves insulin sensitivity. While not being bound by theory, it is believed that the condition of obesity serves to stimulate DPP4 synthesis and secretion into the circulation, by activating the PERK-ATF4 EPv stress pathway in hepatocytes. In turn, DPP4 promotes adipose tissue inflammation by acting on adipose macrophages. Thus, in view of the present data, it is believed that blocking DDP4 secretion from hepatocytes in obese subjects will decrease adipose tissue inflammation and improve insulin sensitivity. Molecular biology

In accordance svith the present invention, there may be numerous tools and techniques within the skill of the art, such as those commonly used in molecular immunology, cellular immunology, pharmacology, and microbiology. See, e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y.; Ausubel et al. eds. (2005) Current Protocols in Molecular Biology. John Wiley and Sons, Inc.: Hoboken, N.J.; Bonifacino et al. eds. (2005) Current Protocols in Cell Biology. John Wiley and Sons, Inc.: Hoboken, N.J.; Coligan et al. eds. (2005) Current Protocols in Immunology, John Wiley and Sons, Inc.: Hoboken, N.J.; Coico et al. eds. (2005) Current Protocols in Microbiology, John Wiley and Sons, Inc.: Hoboken, N.J.; Coligan et al. eds. (2005) Current Protocols in Protein Science, John Wiley and Sons, Inc.: Hoboken, N.J.; and Enna et al. eds. (2005) Current Protocols in Pharmacology, John Wiley and Sons, Inc.: Hoboken, N.J.

"Treating" or "treatment" of a state, disorder or condition includes:

(1) preventing or delaying the appearance of clinical symptoms of the state, disorder, or condition developing in a person who may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical symptoms of the state, disorder or condition; or

(2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof (in case of maintenance treatment) or at least one clinical symptom, sign, or test, thereof; or

(3) relieving the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or sub-clinical symptoms or signs.

The benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician.

A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount. Acceptable excipients, diluents, and carriers for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington: The Science and Practice of Pharmacy. Lippincott Williams & Wilkins (A. R. Gennaro edit. 2005). The choice of pharmaceutical excipient, diluent, and carrier can be selected with regard to the intended route of administration and standard pharmaceutical practice.

An "immune response" refers to the development in the host of a cellular and/or antibody-mediated immune response to a composition or vaccine of interest. Such a response usually consists of the subject producing antibodies, B cells, helper T cells, suppressor T cells, regulatory T cells, and/or cytotoxic T cells directed specifically to an antigen or antigens included in the composition or vaccine of interest.

A "therapeutically effective amount" means the amount of a compound that, when administered to an animal for treating a state, disorder or condition, is sufficient to effect such treatment. The "therapeutically effective amount" will vary depending on the compound, the disease and its severity and the age, weight, physical condition and responsiveness of the animal to be treated.

The compositions of the invention may include a "therapeutically effective amount" or a "prophylactically effective amount" of a compound described herein. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of an antibody or antibody portion may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

While it is possible to use a composition provided by the present invention for therapy as is, it may be preferable to administer it in a pharmaceutical formulation, e.g., in admixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice. Accordingly, in one aspect, the present invention provides a pharmaceutical composition or formulation comprising at least one active composition, or a pharmaceutically acceptable derivative thereof, in association with a pharmaceutically acceptable excipient, diluent and/or carrier. The excipient, diluent and/or carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. The compositions of the invention can be formulated for administration in any convenient way for use in human or veterinary medicine. The invention therefore includes within its scope pharmaceutical compositions comprising a product of the present invention that is adapted for use in human or veterinary medicine.

In a preferred embodiment, the pharmaceutical composition is conveniently administered as an oral formulation. Oral dosage forms are well known in the art and include tablets, caplets, gelcaps, capsules, and medical foods. Tablets, for example, can be made by well-known compression techniques using wet, dry, or fluidized bed granulation methods.

Such oral formulations may be presented for use in a conventional manner with the aid of one or more suitable excipients, diluents, and carriers. Pharmaceutically acceptable excipients assist or make possible the formation of a dosage form for a bioactive material and include diluents, binding agents, lubricants, glidants, disinte grants, coloring agents, and other ingredients. Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, ascorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used. An excipient is pharmaceutically acceptable if, in addition to performing its desired function, it is non-toxic, well tolerated upon ingestion, and does not interfere with absorption of bioactive materials.

Acceptable excipients, diluents, and carriers for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington: The Science and Practice of Pharmacy. Lippincott Williams & Wilkins (A. R. Gennaro edit. 2005). The choice of pharmaceutical excipient, diluent, and carrier can be selected with regard to the intended route of administration and standard pharmaceutical practice.

As used herein, the phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are "generally regarded as safe^*', e.g., that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human. Preferably, as used herein, the term "'pharmaceutically acceptable^"' means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopeias for use in animals, and more particularly in humans.

"Patient" or "subject" refers to mammals and includes human and veterinary subjects. The dosage of the therapeutic formulation will vary widely, depending upon the nature of the disease, the patient's medical history, the frequency of administration, the manner of administration, the clearance of the agent from the host, and the like. The initial dose may be larger, followed by smaller maintenance doses. The dose may be administered as infrequently as weekly or biweekly, or fractionated into smaller doses and administered daily, semi- weekly, etc., to maintain an effective dosage level. In some cases, oral administration will require a higher dose than if administered intravenously. In some cases, topical administration will include application several times a day, as needed, for a number of days or weeks in order to provide an effective topical dose.

The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, olive oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Alternatively, the carrier can be a solid dosage form carrier, including but not limited to one or more of a binder (for compressed pills), a glidant, an encapsulating agent, a flavorant, and a colorant. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin.

This invention will be better understood from the Experimental Details, which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims that follow thereafter.

EXAMPLES Methods:

1. Mouse experiments: Lean mice or DIO mice will be purchased from The Jackson Laboratory. Camk2g-^fl mice were generated as described previously⁸. The mice will be maintained on a 12-h dark-light cycle with high-fat diet (Research diets) feeding for 17 weeks. AAV-TBG- Cr and adeno-ATF4 (0.5-3 x 10 plaque-forming units/mice) will be delivered by tail vein injection and experiments will be commenced after 5-7 days.

2. Real time PGR: First strand cDNA synthesis will be made from RNA, and real time PGR will be performed using primers specific for Mcpl, F480, and Tnfa and SYBR green PGR reagent.

3. Histological analysis: VAT will be fixed in 10 % buffered formalin solution, dehydrated with graded ethanol, cleared with xylene, and embedded in paraffin. Sections (5 mm) will be cut using a Leica RM2125 microtome and stained with hematoxylin and eosin. Images will be obtained using a Nikon Olympus DP25 microscope.

4. SVF assay: SVF cells from obese VAT will be isolated as described²⁶ and cultured with 10 % plasma for 18 h. Total RNA will be isolated and assayed for Mcpl expression using real time PGR.

5. iTRAQ MS: Protein samples (80 mg) will be reconstituted in dissolution buffer and trypsinized for 16 h at 37°C. Tryptic digests will be labeled with four different iTRAQ reagents according to the manufacturer's instructions (iTRAQ Reagents Multiplex kit; Applied Biosystems), pooled together, and subjected to strong cation exchange (SCX) fractionation. Eight fractions will be collected and subjected to MS analysis. The MS data will be analyzed using Proteome Discoverer (Thermo Fisher Scientific, Beta Version 1.2.0.208) and Sequest search algorithm, against the NCBI protein database. Relative quantitation of proteins will be carried out based on the relative intensities of reporter ions released during MS fragmentation of peptides.

6. Statistical analysis: All results are calculated as mean ± SEM. p values are calculated using the Student's t test for normally distributed data and the Mann- Whitney rank sum test for non-normally distributed data. The n number is prospectively estimated using power calculations based on previous date.

The present data (shown in Figures 1A-F) in obese mice with T2D shows that obesity leads to activation of a pathway in hepatocyte (HCs) that induces the synthesis and secretion of DPP4, leading to elevation of DPP4 in the circulation. The liver-derived circulating DDP4 then exacerbates VAT inflammation through effects on adipose tissues macrophages (ATMS). Blocking the DDP4 pathway in HCs markedly blocks VAT inflammation.

In mice and humans with obesity and T2D, activation of the calcium-dependent kinase CaMKII in HCs triggers the PERK-ATF4 branch of the ER stress Unfolded Protein Response (UPR). Blocking this pathway solely in HCs, e.g., by HC-specific knockout of CaMKII, causes a marked improvement in hepatic insulin signaling and an increase in systemic insulin sensitivity, with lowering of FBG and plasma insulin and improvements in ITT and GTT (Ozcan et al. Cell Metabolism 2013).⁸

Blocking this pathway specifically in HCs via HC-specific knockout of CaMKH causes a marked decrease in VAT inflammation, with >75% decrease of macrophages and crown-like structures (CLS), marked lowering of VAT inflammatory cytokines, and decreased monocyte uptake into VAT {See, Fig. 1). This effect was NOT simply due to the improvement in insulin sensitivity.

When ATF4 was restored to the livers of mice with the pathway inhibited, VAT inflammation returned {See, Fig. 2A-D) and when HC specific ATF4 deletion reduced VAT inflammation (See, Fig. 11-A-B), suggesting that this pathway represents a separate and distinct branch of the HC ATF4 pathway. As shown in Fig. 3C, liver inflammation was not affected.

The above data suggest that ATF4 in HCs in obesity induces a circulating "hepatokine" that contributes to VAT inflammation. In support of this concept, the present data shows that plasma from obese mice and humans induces the inflammatory marker Mcpl in ex vivo stromal vascular fraction (SVF) from the VAT obese mice (See, Figs. 3A-C). The factor was heat- inactivated (below), indicating that the factor is a protein and not, for example, lipopolysaccharide. The activity in mouse plasma was much lower in plasma from lean mice and from obese mice in which the ATF4 pathway was genetically disabled in HC— and the plasma factor could be restored by transducing the mutant obese mice with adeno-ATF4 (See, Figs. 7A- E; See also Figs. 4-5.)

The responding cell type in the SVF assay is adipose tissue macrophages (ATMs), not pre-adipocytes. The findings above and below using the SVF assay could be reproduced by using cultured macrophages.

When fractionated on gel-filtration FPLC, the SVF/macrophage-Mepl -inducing factor in plasma appeared as a single peak in the 125-175 kDa range and fit the pattern across our range of mutant mice {See, Figs. 6A-J). Fractions at the peak of activity and nearby fractions that were inactive were subjected to SDS-PAGE, protease fragmentation into peptides, and LC-MS MS shotgun proteomic analysis. One of the few proteins that fit the pattern of activity exactly was DPP4 (See, Fig. 6H). DPP4 activity was higher in obese vs. lean mouse plasma, and, most importantly, SVF-Mcpl -inducing activity in obese mouse plasma was neutralized by adding a DPP4 inhibitor (See, Fig. 61- J). DPP4 activity and ELISA in the plasma of all of the above mouse models fit the pattern of ex-vivo SVF/macrophage-Mcpl -inducing activity in the plasma (which in turn corceiates with in vivo VAT inflammation as above), and it also fit the pattern of Dpp4 mRNA and DPP4 activity in the liver (See, Fig. 7A-E, See ako Figs 8-9). Moreover, Dpp4 mRNA is increased in primary HCs transduced with ATF4.

Primary HCs secrete DPP4 and livers obtained from WT or HC-CaMKIl KO mice or ATF4 restored mice show in vivo evidence that Dpp4 mRNA is spliced into tsvo forms, one full length surface form and another secretory form (See, Figs. 16 A-F). In accordance to this, liver specific deletion of DPP4 lowers circulatory plasma DPP-4 levels which leads to suppression of VAT inflainmation and overall improvement in glucose and insulin tolerance in DIO mice (See, Figs. 12A-G and Figs. 13A-C), suggesting that DPP-4 plays a crucial role during obesity associated VAT inflammation and insulin resistance.

At adipose tissue levels circulatory DPP-4 along with its interacting partners activates PAR2 signaling pathway in SVF cells and macrophages to mediate VAT inflammation (See, Figs. 14A-C and Fig. 15A-C).

Plasma from obese humans had higher SVF-Mcpl -inducing activity, and it fractionated on FPLC similar to mouse plasma (See, Fig. 10A-D).

Elucidating the ATF4-DPP4 pathway in hepatocytes in the setting of obesity.

Based on the present data it is hypothesized that obesity, by activating the PERK- ATF4 ER stress pathway in hepatocytes, stimulates DPP4 synthesis and secretion into the circulation.

Additional studies will verify the role of ATF4 in the DPP4-VAT inflammation pathway in diet-induced obese (DIO) mice by using 4 groups of mice: (1 ) AAV8-shAtf4; (2) Atf4-floxed mice (which we have) crossed with HC-specifie AAV8-TBG-cre mice; and controls (3) AAV8-LacZ and (4) AAV8-TBG-LacZ, respectively. The assay endpoints include: (a) Atf4 and Dpp4 mRNA in liver; (b) SVF-Mcpl -inducing activity, DPP4 activity, and DPP4 ELISA in the plasma: and (c) VAT inflammation: macrophages, crown-like structures (CLS), inflammatory cytokines.

Specific mouse techniques are described above in the methods section, and all protocols have been approved by CUMC IACUC. To use the above experiment as an illustration of the basic principles, we will use 10 male C57BL/6J mice that have been on the DIO diet for 10 wks for each of the 4 groups (40 mice). This n number is suggested by power calculations based on an expected 25-30% coefficient of variation from our previous data and an 80% chance of detecting a 33% difference between control and HC-ATF6- silenced mice in the parameters described below (P = 0.05). To allow room for 2 replications, we will use 120 mice. The mice will be fed high-fat (DIO) diet with 60%' kcal from fat obtained from Research Diets (D12492), and they will be maintained on a 12 hr light-dark cycle. The AAV8 vectors are delivered by tail vein injection at 2-3 X 10¹¹ genome copies/mouse. The mice are monitored frequently for signs of distress by both the researchers in our lab and ICM staff. Euthanasia by CO2 asphyxia followed by cervical dislocation will be used at the end of the experiment and for mice in distress.

In view of the correlation between Dpp4 mRNA in the liver and all plasma and VAT inflammation parameters (described above), it will next be determined whether the increase in Dpp4 with obesity is due to transcription by using pol II and H3K27Ac chromatin immunoprecipitation (ChIP). These studies will be done with primary HCs treated with palmitate, which recapitulates the PERK-ATF4 pathway,⁸ as well as primary HCs treated with thapsigargin as a standard UPR inducer and HCs transfected with ATF4 (see above). (It will be assumed to be due to transcription regulation, but if the above data suggest otherwise, mRNA stability will be explored.) Luciferase reporter constructs driven by the Dpp4 promoter will be made, which will show that when the HCs are transduced with Atf4 (see data above), the luciferase reporter is induced. The Dpp4 promoter does contain putative ATF4 binding sites, and ChIP assays based on this information will be performed to show that ATF4 occupancy of these sites in the various mouse models correlates with hepatic Dpp4 mRNA. If the ATF4 binding sites in the Dpp4 promoter are not revealed by this approach, RNAseq will be used to determine whether any of the sequences immunoprecipitate with anti-ATF4 belong to the promoter of Dpp4 or other genomic regions that could regulate Dpp4 transcription. After knowledge is gained from these studies in HCs, relevance in vivo will be shown by taking advantage of these various mouse models described in the above sections: lean, obese, and liver KO of the ER stress pathway +/- ATF4 restoration. Using 50 mice is anticipated for the primary HC experiments to obtain enough cells for these assays, and, based on similar power calculations as above, the in vivo studies will require 4 models X 10 mice/model x 3 experiments = 120 mice.

It is possible that ATF4 does not directly induce Dpp4 but rather induces a Dpp4 transcriptional activator or represses a Dpp4 repressor. Thus, if ATF4 does not directly bind Dpp4 regulatory elements, RNAseq will be conducted comparing obese mice +/- silenced HC ATF4 (above) and screening the results for transcription factors (or repressors) that match known regulators of Dpp4 or are known to bind cognate cis elements in the Dpp4 promoter. One candidate, the ATF4 target CHOP, has already been ruled out by recent experiments, but other indirect links are possible.

Another possibility is based on studies suggesting a link between circulating 1L-6 and

VAT inflammation. Perhaps IL-6 and DPP4 are in the same pathway. One possibility is that IL-6 functionally interacts with the PERK-ATF4 pathway to induce DPP4, i.e., a 2-hit model such that blocking either ATF4 or IL-6 blocks DPP4 induction. This hypothesis will be tested by immunoneutralizing IL-6 in obese mice via antibody injection (vs. IgG) and testing whether this blocks Dpp4 mRNA in the liver and DPP4 activity/ELISA in the blood. This will require 2 groups X 10 mice/group x 3 experiments = 60 mice. (The converse relationship, i.e., that DPP4 raises circulating IL-6 to cause VAT inflammation, is not supported by the present data that inhibition of DPP4 in plasma blocks SVF and macrophage inflammation.) The role of IL-6 in AT inflammation in obesity has not yet been definitively established in all settings.¹⁰

Once the basic molecular mechanism of transcriptional regulation is known, steps to further elucidate regulation by determining cooperating factors in Dpp4 regulation via through IP and ChIP will be conducted. Causation experiments will begin with siRNA- mediated silencing of the regulatory factors in cultured HCs and then shRNA or Cre-Lox silencing in vivo. The in vivo studies will be focused on the same endpoints listed above for the ATF4 experiments: (a) Atf4 and Dpp4 mRNA in liver; (b) SVF-Mcpl-inducing activity, DPP4 activity, and DPP4 ELISA in the plasma; and (c) VAT inflammation: macrophages, CLS, inflammatory cytokines. The HC experiments will require 50 mice and the in vivo experiments (2 groups in triplicate) will require 60 mice.

These experiments will provide a clear picture, confirming at a molecular level of how obesity leads to the induction of DPP4 in HCs, leading to secretion of DPP4 and DPP4- mediated VAT inflammation.

DPP4 affects inflammatory signaling in adipose macrophages.

DPP4 promotes adipose tissue inflammation by activating inflammatory signaling pathways in adipose macrophages (ATMs).

The VAT inflammatory activity in obese plasma induces inflammation in macrophages in a manner that is blocked by a DPP4 inhibitor. This result could reflect a direct effect of DPP4 on macrophages or a secondary effect due to an action of DPP4 on another protein in the plasma, eg a substrate for its protease activity like GLP1 or G1P. Our current studies show that rDPP4 alone is not able to induce Mcpl mRNA, a marker of VAT inflammation in vivo and ex vivo, but DPP4 isolated from obese plasma by immunoprecipitation does induce Mcpl. rDPP4 combined svifh DPP4 depleted obese plasma is able to induce VAT inflammation suggesting that DPP4 is required to interact with other plasma protein(s) to cause VAT inflammation. Further studies will be done to identify DPP4 partners which along with DPP4 act to induce VAT inflammation during obesity. For studies to date, KR-62436 from Sigma- Aldrich as the DPP4-1 has been used, but any DPP4-I relevant to clinical use and used in previous DIO mouse studies, such as des-fluoro-sitagliptin (DFS) can be used. Based on previous macrophage studies, the concentration will be 25 μΜ.

If IP-DPP4 has a direct effect on macrophages, then the inflammatory signaling hub that is activated will be identified by focusing on several likely candidates from the literature: NFkB, JNK, P38, ERK, PKC, and NLPR3 inflammasome,³'¹¹ '³ with the possibility that more than one of these pathways is activated. The phosphorylation state of P65, the 3 MAPKs, and PKC, and the levels of NLRP3 and IL-Ιβ will be assayed by Western blot in macrophages incubated with IP-DPP4 as above. Positive results will be followed by inhibitor and/or siRNA causation experiments targeting these hubs to determine which is causally important in the ability of IP-DPP4 to induce Mcpl in macrophages. If none of these hubs is involved, other possibilities will be tested using arrays of inflammatory signaling proteins and mRNAs. An unbiased screen using RNAseq and, if necessary to cover post-transcriptional mechanism, shotgun proteomics could be used in additional experiments. Based on these results, upstream signaling will be interrogated, with the ultimate goal to determine the proximal sensor of DPP4. Using the literature as a guide, candidates will be tested first, e.g., activation of protease-activated receptor-2 (PAR2)¹⁴; and inhibition of anti-inflammatory adenosine receptor activation¹⁵, although this activity of DPP4 is membrane bound (CD26) and does not appear to be affected by sitagliptin'⁶, and so is an unlikely candidate. Our data show that pharmacological inhibition of PAR2 reduced obese plasma or IP-DPP4 triggered Mcpl in obese SVF cells and BMDMs. PAR2 siRNA or genetic knock down of PAR2 will be utilized to validate the role of PAR2 in DPP4 triggered VAT inflammation. All positive experiments will be repeated using obese SVF and SVF-derived ATMs to complement the cultured macrophage results, and then in vivo causation experiments will be conducted as described below. If DPP4 has a direct effect on VAT inflammation in obese mice, all efforts will be depletion of the relevant pathway genes in macrophage using available null mice— either floxed mice crossed with macrophage LysMCre or bone marrow transplantation if floxed mice are not available. If no genetic mutants are available, the creation of such mice using conventional means or more likely CR1SPR/Cas9. The goal is to feed the mice a DIO diet and, in comparison with mice without the gene deleted, assess the markers of VAT inflammation outlined above. If DPP4 works indirectly, the first set of in vivo causation experiments will be to neutralize the factor in plasma using available KO mice or antibodies, followed by the macrophage -oriented experiments outlined above.

These experiments will provide confirmation of the mechanisms linking DPP4 to

VAT inflammation. Evaluating the role of the identified factors in VAT inflammation, including links to genetic polymorphisms and mutations could be further explored in obese human subjects. Determining how blocking hepatocyte DPP4 synthesis affects insulin sensitivity in obese mice. Blocking DDP4 secretion from hepatocytes in obese mice, by decreasing adipose tissue inflammation, will improve insulin sensitivity.

Our present data show that liver specific deletion of DPP4 improves insulin sensitivity in obese mice with lowering of VAT inflammatory macrophages, inflammatory cytokine expression and enhanced VAT AKT phosphorylation.

An interesting question related to these studies is how much of the metabolic benefits of DPP4-I are due to suppression of VAT inflammation. Ideally, DFS-treated mice +/- restoration of only the VAT inflammatory pathway would be compared. Additional experiments directly comparing all VAT and metabolic parameters described above in 3 groups of DIO mice: (1) those with the DPP4~macrophage inflammatory pathway inhibited as planned above; (2) DFS-treated; and (3) mouse model from (1) treated with DFS will be carried out. DFS will be added to the DIO diet a 1.1% by weight, based on studies in the literature. A significant but partial improvement in the mice with just the VAT inflammation pathway blocked is predicted, somewhat better improvement with the DFS, and a non- additive effect in the combined model. Assuming 3 groups of mice in triplicate, these experiments will require 90 mice based on the calculations above.

Mechanisms exploring the linking of DPP4-induced VAT inflammation with metabolic disarray, i.e., how blocking DPP4-induced VAT inflammation improves metabolism according to thepredicted findings above, will be carried out. As one example, a recent study by the Shulman group suggested that a key mediator linking VAT inflammation to HGP and insulin resistance might be adipose TG hydrolysis and hepatic acetyl-CoA via the following pathway: adipose macrophage inflammation— > adipose lipolysis— » FA transport and oxidation in liver— » acetyl-CoA→ decrease flux of pyruvate carboxylase (PC)— > lower HGP→ lower stimulation of insulin secretion— > lower insulin resistance.⁹ Therefore, if HGP and glucose output are lower in the present model of decreased DPP4-mediated adipose macrophage inflammation, WT and mutant DIO mice will be assayed for the various markers of this pathway: plasma non-esterified fatty acids and glycerol, liver acetyl-CoA, and liver PC activity.

However, as an example, if blocking DPP4 action on macrophages lowers saturated fatty acids in the blood, i.e., by inhibiting FA lipolysis in adipose tissue, and also lowers insulin-induced p-Akt in liver and or skeletal muscle, the possibility that the DPP4/inflammation-induced saturated fatty acid release from VAT adversely affect insulin signaling in distal tissues, e.g., by triggering inflammatory or ER stress signaling will be considered.¹⁹'²⁰ Alternatively, there are adipokines such as adiponectin that may link DPP4- induced VAT inflammation to insulin resistance in other tissues ¹'²² and so putative adipokines will be assayed in the present model. All in all, the eventual causation experiments should involve 2 groups of mice in triplicate and thus require 60 mice based on the calculations above.

Conclusions

These new discoveries relating to ATF4-mediated induction of DPP4 secretion by HCs in the setting of obesity and the action of DDP4 on VAT inflammation provide a plausible explanation for the observations that DPP4-I have multiple actions of benefit on T2D in humans and that they suppress VAT inflammation in mice. Thus, these findings could provide important mechanistic basis and rationale for additional indications for DPP4-I, e.g., to treat insulin resistance in obese people without overt diabetes (pre-diabetes and metabolic syndrome),⁴ which affects hundreds of millions of people worldwide, hi addition, further elucidation of the mechanisms of liver induction of DPP4 and how it affects ATMs could the form the basis of developing new therapeutics that are more potent than existing DPP4-1 in terms of suppressing AT inflammation and decreasing insulin resistance in obese subjects. Additional studies to understand the mechanisms of liver induction of DPP4 and how it affects adipose macrophages could the form the basis of methods for identifying and screening for new therapeutics that are more potent than existing DPP4-I in terms of suppressing VAT inflammation and decreasing insulin resistance in obese subjects, as well as pre diabetic subjects, and those with metabolic disorder.

References

1. Zhong ., Rao.X., & Rajagopalan,S. An emerging role of dipeptidyl peptidase 4 (DPP4) beyond glucose control: potential implications in cardiovascular disease. Atherosclerosis 226, 305-314 (2013).

2. Sell,H- et al. Adipose dipeptidyl peptidase-4 and obesity: correlation with insulin resistance and depot-specific release from adipose tissue in vivo and in vitro. Diabetes Care 36, 4083-4090 (2013). PMC3836153.

3. Ferrante,A.W., Jr. Obesit -induced inflammation: a metabolic dialogue in the language of inflammation. J. Intern. Med. 262, 408-414 (2007).

4. Goran,M.I. & Alderete,T.L. Targeting adipose tissue inflammation to treat the underlying basis of the metabolic complications of obesity. Nestle. Nutr. Inst. Workshop Ser. 73, 49-60 (2012).

5. Dobrian,A.D. et al. Dipeptidyl peptidase IV inhibitor sitagliptin reduces local inflammation in adipose tissue and in pancreatic islets of obese mice. Am. J. Physiol Endocrinol. Metab 300, E410-E421 (2011). PMC3043624.

6. Shirakawa,J. et al. Diet-induced adipose tissue inflammation and liver steatosis are prevented by DPP-4 inhibition in diabetic mice. Diabetes 60, 1246-1257 (2011 ).

PMC3064098.

7. Shah,Z. et al. Long-term dipeptidyl-peptidase 4 inhibition reduces atherosclerosis and inflammation via effects on monocyte recruitment and chemotaxis. Circulation 124, 2338-

2349 (2011). PMC4224594.

8. Ozcan,L„ BacksJ., 01son,E.N., & Tabas Activation of ealcium/calmodulin-dependent protein kinase II in obesity mediates suppression of hepatic insulin signaling. Cell

Metabolism 18, 803-815 (2013).

9. Perry,R.J. et al. Hepatic acetyl CoA links adipose tissue inflammation to hepatic insulin resistance and type 2 diabetes. Cell 160, 745-758 (2015). pending

10. Kim,J.H., Bachmann,R.A., & Chen,J. Interleukin-6 and insulin resistance. Vitam. Horm. 80, 613-633 (2009). 11. Matsubara,J. et al. A dipeptidyl peptidase -4 inhibitor, des-fluoro-sitagliptin, improves endothelial function and reduces atherosclerotic lesion formation in apolipoprotein E- deficient mice. J. Am. Coll. Cardiol. 59, 265-276 (2012).

12. Zeng,Y. et al. The DPP-4 inhibitor sitagliptin attenuates the progress of atherosclerosis in apolipoprotein-E-knockout mice via. Cardiovasc. Diabetol. 13, 32 (2014). PMC3916068

13. Dai, Y., DaLD., Wang,X., Ding,Z., & Mehta,J.L. DPP-4 inhibitors repress NLRP3 inflammasome and interieukin- 1 beta via GLP- 1 receptor in macrophages through protein kinase C pathway. Cardiovasc. Drugs Ther. 28, 425-432 (2014).

14. Wronkowitz,N. et al. Soluble DPP4 induces inflammation and proliferation of human smooth muscle cells via protease-acdvated receptor 2. Biochim. Biophys. Acta 1842, 1613-

1621 (2014).

15. ZhongJ. el al. A potential role for dendritic cell/macrophage-expressing DPP4 in obesity-induced visceral inflammation. Diabetes 62, 149-157 (2013). PMC3526020.

16. White,P.C, Chamberlain-Shea,H., & de la Morena,M.T. Sitagliptin treatment of patients with type 2 diabetes does not affect CD4+ T-cell activation. J. Diabetes

Complications 24, 209-213 (2010).

19. Wei,Y., Wang,D., Topczewski,F., & Pagliassotti,M.J. Saturated fatty acids induce endoplasmic reticulum stress and apoptosis independently of ceramide in liver cells. Am. J. Physiol Endocrinol. Metab 291, E275-E281 (2006).

20. Ozcan,U. et al. Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes. Science 306, 457-461 (2004).

21. Romacho.T., Elsen.M., Rohrborn,D., & Eckel,J. Adipose tissue and its role in organ crosstalk. Acta Physiol (Oxf) 210, 733-753 (2014).

22. Kirino,Y., Sei.M., Kawazoe.K., Minakuchi,K., & Sato,Y. Plasma dipeptidyl peptidase 4 activity correlates with body mass index and the plasma adiponeclin concentration in healthy young people. Endocr. J. 59, 949-953 (2012).

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

Patents, patent applications, and publications are cited throughout this application, the disclosures of which, particularly, including all disclosed chemical structures, are incorporated herein by reference. Citation of the above publications or documents is not intended as an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. All references cited herein are incorporated by reference to the same extent as if each individual publication, patent application, or patent, was specifically and individually indicated to be incorporated by reference.

The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

Claims

1. A method for decreasing adipose tissue inflammation, comprising blocking DPP4 secretion from hepatocytes in the tissue of a subject in need thereof.

2. The method of claim 1, wherein the subject is suffering from one or more conditions selected from the group consisting of: type 2 diabetes, metabolic syndrome, pre diabetes, obesity, insulin resistance, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, diabetic dysiipidemia, fatty liver disease including non-alcoholic fatty liver disease (NASH), and atherosclerosis.

3. The method of claim 1 , wherein liver-derived DPP4 is reduced .

4. The method of claim 1, wherein blocking DPP4 secretion from hepatocytes improves insulin sensitivity.

5. The method of claim 1 , wherein blocking DPP4 secretion from hepatocytes decreases VAT inflammation.

6. The method of claim 1, wherein the blocking comprises administering an antagonist of DPP4.

7. The method of claim 1 , wherein the blocking comprises hepatic cell-specific genetic knock-dosvn or knockout of DPP4.

8. The method of claim 1, wherein DPP4 blocking is prophylacticaliy administered prior to symptoms normally associated with conditions selected from the group consisting of type 2 diabetes, metabolic syndrome, pre diabetes, obesity , insulin resistance, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, diabetic dysiipidemia, fatty liver disease including non-alcoholic fatty liver disease (NASH), and atherosclerosis.

9. The method of claim 1, further comprising inhibiting PAR2 binding to DPP4 in the tissue.

10. The method of claim 9, wherein inhibiting PAR2 binding comprises contacting the tissue with an antagonist of PAR2.

1 1. A method for decreasing adipose tissue inflammation comprising administering to a subject in need thereof, a compound that blocks DPP4 secretion from hepatocytes.

12. The method of claim 11, wherein the subject is suffering from one or more conditions selected from the group consisting of: type 2 diabetes, metabolic syndrome, pre diabetes, obesity, insulin resistance, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, diabetic dvslipidemia, fatty liver disease including non-alcoholic fatty liver disease (NASH), and atherosclerosis.

13. The method of claim 1 1, wherein the compound is prophylaetically administered prior to symptoms normally associated with conditions selected from the group consisting of type 2 diabetes, metabolic syndrome, pre diabetes, obesity, insulin resistance, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, diabetic dvslipidemia, fatty liver disease including non-alcoholic fatty liver disease (NASH), and atherosclerosis.

14. The method of claim 11, wherein the method further improves insulin sensitivity.

15. The method of claim 1 1, wherein the method further suppresses VAT inflammation.

16. A method for preventing or alleviating obesity comprising administering to a subject in need thereof, a compound that blocks DPP4 secretion from hepatocytes.

17. The method of claim 16, wherein the method further improves insulin sensitivity.

18. The method of claim 16, wherein the method further suppresses VAT inflammation.

19. A method for preventing or alleviating metabolic syndrome comprising administering to a subject in need thereof, a compound that blocks DPP4 secretion from hepatocytes.

20. The method of claim 19, wherein the method further improves insulin sensitivity.

21. The method of claim 19, wherein the method further suppresses VAT inflammation.

22. A method for identifying a compound that blocks DPP4 secretion from hepatocytes comprising: a) incubating a hepatocyte culture with palmitate to induce DPP4 secretion, b) measuring secreted DPP4 in the induced hepatocyte culture, c) contacting and incubating the induced hepatocytes with a test compound, and d) measuring secreted DPP4 in the induced hepatocyte culture following the contacting and incubating step c), wherein a decrease in secreted DPP4 in the induced hepatocvte culture indicates a compound that blocks DPP4 secretion from induced hepatocytes, and wherein the decrease is a level similar to a reference basal DPP4 level observed in medium from non-induced hepatocytes.