WO2018104499A1 - Methods for treating and limiting development of obesity and fatty liver disorders - Google Patents

Abstract

Disclosed herein are methods for treating or limiting development of obesity or treating or limiting development of various fatty liver diseases by administering an inhibitor of apolipoprotein CIII.

Description

Methods for treating and limiting development of obesity and fatty liver disorders Cross reference

This application claims priority to U.S. Provisional Patent Application Nos.

62/431762 filed December 8, 2016 and 62/562695 filed September 25, 2017, each incorporate by reference herein in its entirety.

Background

Obesity and type 2 diabetes (T2D) are closely linked and with the alarming increase of both of these conditions worldwide underscores the importance of identifying better treatment options. A recent survey, where they determined the prevalence of complications among teenagers and young adults that had been diagnosed with type 1 diabetes (T1D) or T2D during childhood or adolescence, revealed that 72% of the patients with T2D and 32% of those with T1D had already developed disease- related complications despite a mean diabetes duration of only 8 years. Furthermore, 90% of the young T2D patients were overweight or obese, while this was seen in 40% of the T1D patients.

Summary

In one aspect are provided methods for treating or limiting development of obesity, comprising administering to an obese subject, or to a subject at risk of obesity, an amount effective of an apolipoprotein CIII (apoCIII) inhibitor to reduce apoCIII expression and/or activity to control levels, thereby reducing body weight or reducing the rate of body weight increase in the obese subject or the subject at risk of obesity. In one embodiment, the subject is obese. In another embodiment, the subject is at risk of obesity, such as having a parent that is obese, having a sedentary lifestyle, consuming a high fat diet, having Prader-Willi syndrome or Cushing's syndrome, taking medications that lead to weight gain, including but not limited to antidepressants, anti-seizure medications, diabetes medications, antipsychotic medications, steroids and beta blockers, being age 55 or older (55, 60, 65, 70 years of age, or older), being sleep deprived (including but not limited to having sleep apnea), and/or quitting smoking.

In another embodiment, the subject is on a high fat diet, wherein the apoCIII inhibitor limits the diet-induced increase of apoCIII in the subject. In another embodiment, the subject has diabetes, such as type 2 diabetes or type 1 diabetes. In a further embodiment, the treating comprises normalizing glucose tolerance and/or limiting fatty liver disease. In another embodiment, reducing body weight or reducing the rate of body weight increase comprises reducing fat levels in the subject, or reducing the increase in fat levels in the subject.

In a further embodiment, the apoCIII inhibitor is selected from the group consisting of anti-apoCIII antibody, anti-apoCIII aptamer, apoCIII small interfering RNA, apoCIII small internally segmented interfering RNA, apoCIII short hairpin RNA, apoCIII microRNA, and apoCIII antisense oligonucleotides. In another embodiment, the apoCIII inhibitor comprises or consists of ASO-ISIS 353982 (AS02).

In a second aspect are provided methods for treating fatty liver disease (FLD), nonalcoholic fatty liver (NAFL), and/or Non-alcoholic fatty liver disease (NAFLD) (such as nonalcoholic steatohepatitis (NASH)), comprising administering to a subject having FLD, NAFL, and/or NAFLD (such as NASH) an amount effective of an apoCIII inhibitor to treat FLD, NAFL, and/or NAFLD (such as NASH). In a third aspect are provided methods for limiting development of FLD, NAFL, and/or NAFLD (such as NASH), comprising administering to a subject at risk of FLD, NAFL, and/or NAFLD (such as NASH) an amount effective of an apoCIII inhibitor to limit development of FLD, NAFL, and/or NAFLD (such as NASH). In one embodiment, the method is for treating or limiting development of NAFLD (such as NASH). In another embodiment, the apoCIII inhibitor reduces apoCIII expression and/or activity in the subject to control levels. In a further embodiment, the subject has a risk factor for fatty liver disease selected from the group consisting of diabetes, obesity, insulin-resistance, a patatin-like phospholipase domain-containing 3 (PNPLA3) 148 MM variant, single-nucleotide polymorphisms (SNPs) T455C and C482T in apolipoprotein CIII (APOC3), hypertension, dyslipidemia, abetalipoproteinemia, , glycogen storage diseases, Weber-Christian disease, acute fatty liver of pregnancy, lipodystrophy, malnutrition, severe weight loss, refeeding syndrome, jejunoileal bypass, gastric bypass, jejunal diverticulosis with bacterial overgrowth, exposure to drugs or toxins (including but not limited to exposure to amiodarone, methotrexate, diltiazem, expired tetracycline, highly active antiretroviral therapy, glucocorticoids, tamoxifen- and environmental hepatotoxins (e.g., phosphorus, mushroom poisoning)), alcoholism, celiac disease, inflammatory bowel disease, HIV infection, hepatitis C infection, disparate levels of serum alanine transaminase and aspartate transaminase in the liver, and alpha 1 -antitrypsin deficiency. In another embodiment, the subject is on a high fat diet, wherein the apoCIII inhibitor limits the diet-induced increase of apoCIII in the subject. In a further embodiment, the apoCIII inhibitor is selected from the group consisting of anti- apoCIII antibody, anti-apoCIII aptamer, apoCIII small interfering RNA, apoCIII small internally segmented interfering RNA, apoCIII short hairpin RNA, apoCIII microRNA, and apoCIII antisense oligonucleotides. In another embodiment, the apoCIII inhibitor comprises or consists of ASO-ISIS 353982 (AS02). In other embodiments, the method comprises one or more of decreasing liver lipid/fat content, decreasing liver inflammation, reducing the rate of increase in liver lipid/fat content, reducing the rate of increase of liver inflammation, limiting any increase/reducing incidence of cardiovascular disease and or type 2 diabetes; reducing the histologically defined NAS activity score >/= 2 (see below) with no worsening of liver fibrosis; reducing liver fibrosis, reducing the rate of increase in liver fibrosis, reducing liver failure, and/or slowing the progression to liver failure.

In a fourth aspect are provided methods for identifying a compound for treating obesity, limiting development of obesity, treating FLD, NAFL, and/or NAFLD (such as NASH), or limiting development of FLD, NAFL, and/or NAFLD (such as NASH), comprising

(a) treating a first test animal with a high fat diet;

(b) treating a second test animal with (i) the high fat diet, (ii) a test compound; and

(c) determining plasma apoCIII levels and/or activity in the first test animal and the second test animal;

wherein test compounds that reduce plasma apoCIII levels and/or activity in the second test animal compared to plasma apoCIII levels and/or activity in the first test animal are candidate compounds for treating or limiting development of obesity or FLD, NAFL, and/or NAFLD (such as NASH).

In one embodiment, the method further comprises

(d) treating a third test animal with a non-high fat diet and determining plasma apoCIII levels and/or activity in the third test animal,

wherein test compounds that lead to plasma apoCIII levels and/or activity in the second test animal similar to plasma apoCIII levels and/or activity in the third test animal are candidate compounds for treating or limiting development of obesity or FLD, NAFL, and/or NAFLD (such as NASH). In another embodiment, the candidate compounds are candidate compounds for treating obesity or FLD, NAFL, and/or NAFLD (such as NASH) in a diabetic subject. In a further embodiment, the test animals are mice. In a further embodiment, the high fat diet comprises on a caloric basis between about 40% and about 80% fat, about 10% to about 30% carbohydrate, and about 10% to about 30% protein. In another embodiment, the high fat diet comprises on a caloric basis about 60% fat, about 20% carbohydrate, and about 20% protein. In one embodiment, the non-high fat diet comprises, on a caloric basis, about 5% to about 15% fat, about 50% to about 75% carbohydrates, and about 15% to about 40% protein. In a further embodiment, the non-high fat diet comprises, on a caloric basis, about 11.4% fat, about 63% carbohydrates, and about 26% protein.

Description of the Figures

Figure 1. Lowering apoCIII reverses diet-induced obesity, insulin resistance and IGT in mice on HFD. a, Body weight (BW) during the time course of the study (n = 6-8). Black arrow indicates the time point when the ASO targeting apoCIII started to be administered in untreated mice on HFD for 10 weeks (see methods), b, Representative picture of ASO+HFD (left) and Scr+HFD (right) mice at the end of the study, c, Representative immunoblot and densitometry analysis of circulating apoCIII in albumin-depleted plasma samples from

Control, Scr+HFD and ASO+HFD mice at the end of the study (n = 4 in triplicate), d, Blood glucose levels during IPITT (left) and area under the curve (AUC) for the IPITT in 12-h overnight fasted mice (n = 6-7). e, Blood glucose levels during IPGTT (left) and AUC for the IPGTT (right) in 12-h overnight fasted mice (n = 6-8). f, Triglyceride and g, cholesterol lipid profiles measured in VLDL-, IDL-, LDL- and HDL-eluted plasma fractions from Scr+HFD and ASO+HFD mice at the end of the study (n = 7). h, Hematoxylin and eosin (H&E) imaging of liver sections from Control, Scr+HFD and ASO+HFD mice at the end of the study (n=3 in triplicate). Scale bar, 100 μιη. i, Hepatic triglycerides content analyzed by spectrophotometry in liver samples from Control, Scr+HFD and ASO+HFD (n = 6-7) mice. All data are mean + standard error of the mean (s.e.m.) and analyzed by one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test. *P < 0.05 Scr+HFD versus Control mice and #P < 0.05 ASO+HFD versus Scr+HFD mice.

Figure 2. Four weeks of ASO treatment improves insulin sensitivity and glucose tolerance despite consumption of a HFD. a, Body weight (BW) during the time course of the study (n = 6). Black arrow at 10 weeks of diet intervention indicates the time point when the HFD-fed mice were divided into two groups, one receiving the Scr ASO and the other one receiving the active ASO against apoCIII for 4 additional weeks until completion of the study. Mice on control diet were given saline (see methods), b, Whole-body fat mass and c, lean mass measured by NMR in Scr+HFD and ASO+HFD mice at the end of the study (n = 8). d, Representative immunoblot and densitometry analysis of circulating apoCIII in albumin- depleted plasma samples from Control, Scr+HFD and ASO+HFD mice at the end of the study (n = 4 in triplicate), e, Blood glucose levels during IPITT (left) and area under the curve (AUC) for the IPITT in 6-h fasted mice (n = 6). f, Blood glucose levels during IPGTT (left) and AUC for the IPGTT (right) in 6-h fasted mice (n = 6). g-h, Metabolic parameters monitored during 2 consecutive days in Scr+HFD and ASO+HFD mice using a

comprehensive Lab Animal Monitoring System (CLAMS) (n = 8). g, Averaged V0₂ (left) and representative traces for VO2 (right), h, Averaged VCO2 (left) and representative traces for VCO2 (right), i, Averaged daily food intake (left) and cumulative food intake (right), j, Averaged energy expenditure (left) and representative traces for energy expenditure. All data are mean + s.e.m. One-way ANOVA followed by Tukey's post-hoc test for a-f; Non- parametric Friedman test followed by Dunn's multiple comparison test for g-h. *P < 0.05 Scr+HFD versus Control mice and #P < 0.05 ASO+HFD versus Scr+HFD mice.

Figure 3. Four weeks of ASO treatment improves lipid profiles and liver lipid catabolism in HFD-fed mice, a, b, Plasma lipid profiles in mice subjected to a 10-week HFD intervention and thereafter injected with either a Scr ASO or an active ASO against apoCIII for 4 additional weeks until completion of the study (see methods), a, Triglyceride and b, cholesterol lipid profiles measured in VLDL-, IDL-, LDL- and HDL-eluted plasma fractions from Scr+HFD and ASO+HFD mice at the end of the study (n = 4). c, Oil red Ό' (ORO) imaging of liver sections from Control, Scr+HFD and ASO+HFD mice at the end of the study (n = 3 in triplicate). Scale bar, 100 μιη. d, Hepatic triglycerides content analyzed by spectrophotometry in liver samples from Control, Scr+HFD and ASO+HFD mice at the end of the study (n = 4). e-m, Analysis of gene expression by quantitative real-time PCR (qRT- PCR) in liver tissue from Control, Scr+HFD and ASO+HFD mice at the end of the study (n = 4). mRNA transcript levels of e, apoCIII; f, Lipc; g, Ppara; h, Rxr; i, Cptl; j, Ldlr; k, Srbl; 1, Cd36; and m, ATP-binding cassette sub-family G member 5 (Abcg5, left) and 8 (Abcg8, right) were quantified and represented as fold increase/decrease relative to Control mice (dashed line), β-actin was used as a housekeeping gene. All data are mean + s.e.m. and analyzed by one-way ANOVA followed by Tukey's post-hoc test. *P < 0.05 Scr+HFD versus Control mice and #P < 0.05 ASO+HFD versus Scr+HFD mice.

Figure 4. Lowering apoCIII with ASO treatment prevents diet-induced obesity, insulin resistance and IGT. a, Body weight (BW) during the time course of the study (n = 6). Black arrow indicates that the treatment with either the Scr ASO or the active ASO against apoCIII started simultaneously with the HFD intervention. Mice on control diet were given saline (see methods), b Whole -body fat mass and c, lean mass measured by NMR in Scr+HFD and ASO+HFD mice at the end of the study (n = 7-8).

Figure 5. ASO treatment reducing apoCIII prevents dyslipidemia and ectopic fat accumulation by inducing liver lipid catabolism a, b, Plasma lipid profiles in mice injected with either the Scr ASO or the active ASO against apoCIII and simultaneously fed a HFD (see methods), a, Triglyceride and b, cholesterol lipid profiles measured in VLDL-, IDL-, LDL- and HDL-eluted plasma fractions from Scr+HFD and ASO+HFD mice at the end of the study (n = 6). c, ORO imaging of liver sections from Control, Scr+HFD and ASO+HFD mice at the end of the study (n = 3 in triplicate). Scale bar, 100 μιη. d, Hepatic triglycerides content analyzed by spectrophotometry in liver samples from Control, Scr+HFD and ASO+HFD mice at the end of the study (n = 5). e-m, Analysis of gene expression by qRT-PCR in liver tissue from Control, Scr+HFD and ASO+HFD mice at the end of the study (n = 4). mRNA transcript levels of e, apoCIII; f, Lipc; g, Ppara; h, Rxr; i, Cptl; j, Ldlr; k, Srbl; 1, Cd36; m, Abcg5 (left) and Abcg8 (right) were quantified and represented as fold increase/decrease relative to Control mice (dashed line), β-actin was used as a housekeeping gene. All data are mean + s.e.m. and analyzed by one-way ANOVA followed by Tukey's post-hoc test. *P < 0.05 Scr+HFD versus Control mice and #P < 0.05 ASO+HFD versus Scr+HFD mice.

Detailed Description

All references cited are herein incorporated by reference in their entirety. Within this application, unless otherwise stated, the techniques utilized may be found in any of several well-known references such as: Molecular Cloning: A Laboratory Manual (Sambrook, et al.,

1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, CA), "Guide to Protein Purification" in Methods in Enzymology (M.P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al.

1990. Academic Press, San Diego, CA), Culture of Animal Cells: A Manual of Basic

Technique, 2^nd Ed. (R.I. Freshney. 1987. Liss, Inc. New York, NY), Gene Transfer and Expression Protocols, pp. 109-128, ed. E.J. Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin, TX).

As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. "And" as used herein is interchangeably used with "or" unless expressly stated otherwise. All embodiments of any aspect disclosed herein can be used in combination, unless the context clearly dictates otherwise.

In a first aspect is provided methods for treating or limiting development of obesity, comprising administering to an obese subject or a subject at risk of obesity an amount effective of an apolipoprotein CIII (apoCIII) inhibitor to reduce apoCIII expression and/or activity to control levels, thereby reducing body weight or reducing the rate of body weight increase in the obese subject or the subject at risk of obesity.

The inventors have surprisingly discovered that reducing apoCIII expression and/or activity to control levels can both limit and reverse the deleterious effects of diet in obese subjects or subjects at risk of obesity, thus reducing body weight or reducing the rate of body weight increase in the obese subject or the subject at risk of obesity. Targeting apoCIII may thus be central to avoiding the negative consequences of an unhealthy diet, for example, in association with diabesity.

The obese subject is a subject that has a body mass index (BMI) of 30 and above, where BMI is a person' s height in kilograms divided by their height in meters squared. The subject at risk of obesity is one that has one or more risk factors for developing obesity, including but not limited to having a parent that is obese, having a sedentary lifestyle, consuming a high fat diet, having Prader-Willi syndrome or Cushing's syndrome, taking medications that lead to weight gain, including but not limited to antidepressants, anti-seizure medications, diabetes medications, antipsychotic medications, steroids and beta blockers, being age 55 or older (55, 60, 65, 70 years of age, or older), being sleep deprived (including but not limited to having sleep apnea), and/or quitting smoking.

As used herein, "treating obesity" means accomplishing one or more of the following: (a) reducing the severity of obesity or obesity complications; (b) limiting or preventing development of obesity complications; (c) inhibiting worsening of obesity complications or of symptoms characteristic of obesity; (d) limiting or preventing recurrence of obesity complications or of symptoms characteristic of obesity; (e) limiting or preventing recurrence of obesity complications or of symptoms characteristic of obesity in patients that were previously symptomatic; (f) reducing body weight or reducing the rate of body weigh increase; (g) normalizing glucose tolerance, (h) reducing liver steatosis; and/or (i) reducing fat levels in the subject, or reducing the increase in fat levels in the subject.

As used herein, "limiting development of obesity" means accomplishing one or more of the following: (a) slowing or preventing the onset of obesity or obesity complications; (b) reducing body weight or reducing the rate of body weigh increase; (c) normalizing glucose tolerance, (d) reducing liver steatosis; and/or (e) reducing fat levels in the subject, or reducing the increase in fat levels in the subject.

In another aspect is provided methods for treating or limiting development of fatty liver disease (FLD ), non-alcoholic fatty liver ( NAFL). non-alcoholic fatty liver disease (NAFLD), such as non-alcoholic steatohepatitis (NASH), comprising administering to a subject having FLD, NAFL, and/or NAFLD (such as NASH) or at risk of FLD, NAFL, and/or NAFLD (such as NASH) an amount effective of an apoCIII inhibitor to treat or limit development of FLD, NAFL, and/or NAFLD (such as NASH).

The inventors have surprisingly discovered that reducing apoCIII expression and/or activity can both treat and limit development of FLD, NAFL, and/or NAFLD (such as NASH).

As used herein, "Fatty liver disease" is a condition associated with intracytoplasmic accumulation of large vacuoles of triglyceride fat in liver cells via steatosis (i.e., abnormal retention of lipids within a cell). In one embodiment, the fatty liver disease may be steatosis (non-alcoholic fatty liver (NAFL)). In another embodiment, fatty liver disease may nonalcoholic fatty liver disease (NAFLD), including but not limited to non-alcoholic

steatohepatitis (NASH), the most extreme form of NAFLD. NAFLD is one of the types of fatty liver which occurs when fat is deposited (steatosis) in the liver due to causes other than excessive alcohol use. By definition, alcohol consumption of over 20 g/day (about 25 ml/day of net ethanol) excludes the condition. The fatty liver disease, such as NAFLD, may involve worsening liver fibrosis, which can progress to development of cirrhosis and increased risk of end stage liver disease. The subject at risk of FLD, NAFL, and/or NAFLD (such as NASH) is one that has one or more risk factors for developing FLD, NAFL, and/or NAFLD, including but not limited to diabetes, obesity, insulin-resistance, a patatin-like phospholipase domain- containing 3 (PNPLA3) 148MM variant, single-nucleotide polymorphisms (SNPs) T455C and C482T in apolipoprotein CIII (APOC3), hypertension, dyslipidemia,

abetalipoproteinemia, , glycogen storage diseases, Weber-Christian disease, acute fatty liver of pregnancy, lipodystrophy, malnutrition, severe weight loss, refeeding syndrome, jejunoileal bypass, gastric bypass, jejunal diverticulosis with bacterial overgrowth, exposure to drugs or toxins (including but not limited to exposure to amiodarone, methotrexate, diltiazem, expired tetracycline, highly active antiretroviral therapy, glucocorticoids, tamoxifen- and

environmental hepatotoxins (e.g., phosphorus, mushroom poisoning)), alcoholism, celiac disease, inflammatory bowel disease, HIV infection, hepatitis C infection, disparate levels of serum alanine transaminase and aspartate transaminase in the liver, and alpha 1 -antitrypsin deficiency.

As used herein, "treating FLD, NAFL, and/or NAFLD (such as NASH)" means accomplishing one or more of the following: (a) reducing the severity of FLD, NAFL, and/or NAFLD (such as NASH) or FLD, NAFL, and/or NAFLD (such as NASH) complications; (b) limiting or preventing development of FLD, NAFL, and/or NAFLD (such as NASH) complications; (c) inhibiting worsening of FLD, NAFL, and/or NAFLD (such as NASH) complications or of symptoms characteristic of FLD, NAFL, and/or NAFLD (such as NASH); (d) limiting or preventing recurrence of FLD, NAFL, and/or NAFLD (such as NASH) complications or of symptoms characteristic of FLD, NAFL, and/or NAFLD (such as NASH); (e) limiting or preventing recurrence of FLD, NAFL, and/or NAFLD (such as NASH) complications or of symptoms characteristic of FLD, NAFL, and/or NAFLD (such as NASH) in patients that were previously symptomatic;

As used herein, "limiting development of FLD, NAFL, and/or NAFLD (such as

NASH)" means slowing or preventing the onset of FLD, NAFL, and/or NAFLD (such as NASH) or FLD, NAFL, and/or NAFLD (such as NASH) complications.

In various embodiments, the methods comprise one or more of decreasing liver lipid/fat content, decreasing liver inflammation, reducing the rate of increase in liver lipid/fat content, reducing the rate of increase of liver inflammation, limiting any i nc rease/red uc i ng incidence of cardiovascular disease and or type 2 diabetes: reducing the histologically defined NAS activity score >/= 2 (see below) with no worsening of liver fibrosis; reducing liver fibrosis, reducing the rate of increase in liver fibrosis, reducing liver failu e, and/or slowing the progression to liver failure.

Histological Scoring System for Nonalcoholic Fatty Liver Disease Components of NAFLD Activity Score (NAS) and Fibrosis Staging

(Nonalcoholic Steatohepatitis Clinical Research Network)

NAS Components (see scoring interpretation)

Item Score Extent Definition and Comment

Steatosis 0 <5% Refers to amount of surface area involved by steatosis as

evaluated on low to medium power examination; minimal steatosis (<5%) receives a score of 0 to avoid giving excess weight to biopsies with very little fatty change

1 5-33%

2 >33-66%

3 >66%

Lobular 0 No foci Acidophil bodies are not included in this assessment, nor is Inflammation portal inflammation

1 <2 foci/200x

2 2-4 foci/200x

3 >4 foci/200x

Hepatocyte 0 None

Ballooning 1 Few balloon The term "few" means rare but definite ballooned hepatocytes as cells well as cases that are diagnostically borderline

2 Many Most cases with prominent ballooning also had Mallory's hyalin, cells/prominent but Mallory's hyaline is not scored separately for the NAS ballooning

Fibrosis Stage (Evaluated separately from NAS)

Fibrosis 0 None

1 Perisinusoidal or

periportal

1A Mild, zone 3, "delicate" fibrosis

perisinusoidal

IB Moderate, zone "dense" fibrosis

3, perisinusoidal

1C Portal/periportal This category is included to accommodate cases with portal and/or peri portal fibrosis without accompanying

pericellular/perisinusoidal fibrosis

2 Perisinusoidal

and

portal/periportal

3 Bridging fibrosis

4 Cirrhosis

Total NAS score represents the sum of scores for steatosis, lobular inflammation, and ballooning, and ranges from 0-8. Diagnosis of NASH (or, alternatively, fatty liver not diagnostic of NASH) should be made first, then NAS is used to grade activity. In the reference study, NAS scores of 0-2 occurred in cases largely considered not diagnostic of NASH, scores of 3-4 were evenly divided among those considered not diagnostic, borderline, or positive for NASH. Scores of 5-8 occurred in cases that were largely considered diagnostic of NASH

In another embodiment or each of the above aspects, the apoCIII inhibitor reduces apoCIII expression and/or activity in the subject to control levels. Any suitable control level can be used, including apoCIII expression and/or activity levels from a subject or population of subjects known not to be obese, not having known risk factors for developing obesity, or known not to have FLD or risk factors for FLD. In one embodiment, reducing apoCIII expression to control levels means to reduce apoCIII expression and/or activity in the obese subject, the subject at risk of obesity, the subject having FLD, or the subject at risk of FLD between 10%-90% compared to apoCIII expression and/or activity prior to treatment. In various further embodiments, reducing apoCIII expression and/or activity to control levels means to reduce apoCIII expression and/or activity in the obese subject, the subject at risk of obesity, the subject having FLD, or the subject at risk of FLD between about 10 -80 , 10%-70%, 10%-60%, 10%-50%, 10%-40%, 10%-30%, 10%-20%, 20 -90 , 20 -80 , 20 -70 , 20 -60 , 20 -50 , 20 -40 , 20 -30 , 30 -90 , 30 -80 , 30 -70 , 30 -60 , 30 -50 , 30 -40 , 40 -90 , 40 -80 , 40 -70 , 40 -60 , 40 -50 , 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-90%, 60%-80%, 60%-70%, 70%-90%, 70%-80%, 80%-90%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% compared to apoCIII expression and/or activity prior to treatment. In one specific embodiment, reducing apoCIII expression and/or activity to control levels means to reduce apoCIII expression and/or activity in the obese subject, the subject at risk of obesity, the subject having FLD, or the subject at risk of FLD, between about 30% to about 80% compared to apoCIII expression and/or activity prior to treatment. In a preferred embodiment, apoCIII expression and/or activity is detected in blood or serum samples. In one embodiment to evaluate the levels and/or activity of apoCIII in sera, albumin is removed from serum samples using standard techniques, such as via use of Montage Albumin Deplete Kit (Millipore) or AlbuSorb™ (Biotech Support Group). The collected sera samples can then be freeze-dried overnight and run on sep-Pak™ CI 8. The eluted proteins can be freeze-dried and thereafter dissolved in 100 μΐ. 0.1% TFA and run on an ACE C18 10- x 0.21-cm column 20- 60%, and the area under the curve, where apoCIII elutes, evaluated. ApoCIII can be identified using any suitable technique, including but not limited to MALDI mass spectrometry.

As used herein, an "inhibitor" of ApoCIII expression and/or activity includes compounds that reduce the transcription of ApoCIII DNA into RNA, compounds that reduce translation of the ApoCIII RNA into protein, and compounds that reduce activity of ApoCIII protein. The inhibitor is a partial inhibitor, such that the expression and/or activity ApoCIII reduced to control levels, as noted above, and as exemplified herein. Such inhibitors are selected from the group consisting of antisense oligonucleotides directed against the ApoCIII DNA, or mRNA; small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), microRNAs (miRNA) or small internally segmented interfering RNAs (sisiRNA) directed against the ApoCIII, protein, DNA, or mRNA, ApoCIII antibodies, aptamers that bind to ApoCIII, and any other chemical or biological compound that can interfere with ApoCIII expression and/or activity. Based on the present disclosure in light of the level of skill in the art, those of skill in the art can readily identify other apoCIII inhibitors that are partial inhibitors.

In various embodiments, the apoCIII inhibitor is selected from the group consisting of apoCIII small interfering RNA, apoCIII small internally segmented interfering RNA, apoCIII short hairpin RNA, apoCIII microRNA, apoCIII antisense oligonucleotides, ApoCIII antibodies, and aptamers that bind to ApoCIII. In each aspect, the subject may be any subject that can benefit from the methods of treatment disclosed herein, including humans, cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses, chickens, and so on. Most preferably, the subject is human. In another specific embodiment, the subject has diabetes, such as type 1 or 2 diabetes. In another embodiment, the subject is on a high fat diet (i.e.: a diet rich in fats, especially saturated fats); in this embodiment, the apoCIII inhibitor limits the diet-induced increase of apoCIII. In this embodiment, the high fat diet may be one that provides on a caloric basis at least 30% of energy as fat (i.e.: at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more). In one embodiment, the high fat diet comprises on a caloric basis between about 40% and about 80% fat, about 10% to about 30% carbohydrate, and about 10% to about 30% protein. In another embodiment, the high fat diet comprises on a caloric basis about 60% fat, about 20% carbohydrate, and about 20% protein.

The methods disclosed herein can significantly reduce the risk of obesity and/or FLD, NAFL, and/or NAFLD (such as NASH) complications, including but not limited to limiting development of, limiting the rate of development of, and/or limiting severity of coronary heart disease, high blood pressure, stroke, sleep apnea, gallstones, and osteoarthritis in the obese subject, and limiting the rate of development of, and/or reducing hepatocyte necrosis, hepatic fibrosis, liver lipid/fat content, liver inflammation, reducing the rate of increase in liver lipid/fat content, reducing the rate of increase of liver inflammation, limiting any

increase/reducing incidence of cardiovascular disease and or type 2 diabetes; reducing the histologically defined NAS activity score >/= 2 (see below) with no worsening of liver fibrosis; reducing liver fibrosis, reducing the rate of increase in liver fibrosis, reducing liver failure, slowing the progression to liver failure, and reducing the rate of or progression to cirrhosis.

In one embodiment, the apoCIII inhibitor is an antisense oligonucleotide. Any suitable apoCIII antisense oligo nucleotide inhibitor can be used that results in the partial inhibition of apoCIII expression and/or activity to control levels as defined above. Exemplary such antisense oligonucleotides include (but are not limited to) the following (see US

8530439):

# REGION SEQUENCE SEQ ID

340982 Coding TGAACTTATCAGTGAACTTG 1

340987 Coding TCAGGGCCAGACTCCCAGAG 2

340988 Coding TTGGTGTTGTTAGTTGGTCC 3 # REGION SEQUENCE SEQ ID

340991 Coding TTGGTGTTGTTAGTTGGTCC 4

353932 Coding AGAGCCACGAGGGCCACGAT 5

353933 Coding AGAGGCCAGGAGAGCCACGA 6

353934 Coding CAGCTCGGGCAGAGGCCAGG 7

353935 Coding TCTCCCTCATCAGCTCGGGC 8

353936 Coding GCCCAGCAGCAAGGATCCCT 9

353937 Coding CCTGCATAGAGCCCAGCAGC 10

353938 Coding TCCATGTAGCCCTGCATAGA 11

353940 Coding GGACCGTCTTGGAGGCTTGT 12

353941 Coding AGTGCATCCTGGACCGTCTT 13

353942 Coding CATGCTGCTTAGTGCATCCT 14

353943 Coding CAGACTCCTGCATGCTGCTT 15

353944 Coding ACAGCTATATCAGACTCCTG 16

353945 Coding CTGGCCACCACAGCTATATC 17

353946 Coding AAGCGATTGTCCATCCAGCC 18

353949 Coding TGCTCCAGTAGCCTTTCAGG 19

353950 Coding GAACTTGCTCCAGTAGCCTT 20

353951 Coding CAGTGAACTTGCTCCAGTAG 21

353952 Coding CTTATCAGTGAACTTGCTCC 22

353953 Coding CCAGTGAACTTATCAGTGAA 23

353954 Coding GAGGCCAGTGAACTTATCAG 24

353955 Coding CCAGAGGCCAGTGAACTTAT 25

353956 Coding GACTCCCAGAGGCCAGTGAA 26

353957 Coding GGCCAGACTCCCAGAGGCCA 27

353958 Coding AGTTGGTCCTCAGGGCCAGA 28

353959 Coding GTTAGTTGGTCCTCAGGGCC 29

353960 Coding TGTTGTTAGTTGGTCCTCAG 30

353961 Coding AGAGTTGGTGTTGTTAGTTG 31

353962 Coding GCTCAAGAGTTGGTGTTGTT 32

353963 Coding CACGGCTCAAGAGTTGGTGT 33

353964 Stop Codon GTCTCACGGCTCAAGAGTTG 34

353966 Stop Codon GAACATGGAGGTCTCACGGC 35

353967 Stop Codon TCTGGAACATGGAGGTCTCA 36

353968 3'UTR CACATCTGGAACATGGAGGT 37

353969 3'UTR CAGACACATCTGGAACATGG 38

353970 3'UTR TGGCCAGACACATCTGGAAC 39

353972 3'UTR AGGATAGATGGCCAGACACA 40

353973 3'UTR CAGCAGGATAGATGGCCAGA 41

353974 3'UTR GAGGCAGCAGGATAGATGGC 42 # REGION SEQUENCE SEQ ID

353975 3'UTR TTCGGAGGCAGCAGGATAGA 43

353976 3'UTR AACCTTCGGAGGCAGCAGGA 44

353977 3' UTR GAGCAACCTTCGGAGGCAGC 45

353978 3'UTR CTTAGAGCAACCTTCGGAGG 46

353979 3' UTR TCCCCTTAGAGCAACCTTCG 47

353980 3'UTR ACTTTCCCCTTAGAGCAACC 48

353981 3'UTR ATATACTTTCCCCTTAGAGC 49

353982 3'UTR GAGAATATACTTTCCCCTTA 50

353983 3'UTR GCATGAGAATATACTTTCCC 51

353984 3'UTR AAAGGCATGAGAATATACTT 52

353985 3'UTR GGATAAAGGCATGAGAATAT 53

353986 3'UTR GGAGGGATAAAGGCATGAGA 54

353987 3'UTR GCATGTTTAGGTGAGGTCTG 55

353988 3'UTR GACAGCATGTTTAGGTGAGG 56

353990 3'UTR TTATTTGGGACAGCATGTTT 57

353991 3'UTR GCTTTTATTTGGGACAGCAT 58

353992 3'UTR TCCCAGCTTTTATTTGGGAC 59

SEQ ID

# REGION SEQUENCE

NO

CACGATGAGGAGCATTCGGG 60 AGGGCCACGATGAGGAGCAT 61 CCACGAGGGCCACGATGAGG 62 GAGAGCCACGAGGGCCACGA 63 GCCAGGAGAGCCACGAGGGC 64 CAGAGGCCAGGAGAGCCACG 65 TCGGGCAGAGGCCAGGAGAG 66 TCAGCTCGGGCAGAGGCCAG 67 CCTCATCAGCTCGGGCAGAG 68 CTCTCCCTCATCAGCTCGGG 69 GATCCCTCTCCCTCATCAGC 70 GCAAGGATCCCTCTCCCTCA 71 CAGCAGCAAGGATCCCTCTC 72 GAGCCCAGCAGCAAGGATCC 73 GCATAGAGCCCAGCAGCAAG 74 GCCCTGCATAGAGCCCAGCA 75 ATGTAGCCCTGCATAGAGCC 76 GTTCCATGTAGCCCTGCATA 77 GGCTTGTTCCATGTAGCCCT 78 # REGION SEQUENCE

NO

340956 Coding TTGGAGGCTTGTTCCATGTA 79

340957 Coding CCGTCTTGGAGGCTTGTTCC 80

340958 Coding CTGGACCGTCTTGGAGGCTT 81

340959 Coding GCATCCTGGACCGTCTTGGA 82

340960 Coding TTAGTGCATCCTGGACCGTC 83

340961 Coding GCTGCTTAGTGCATCCTGGA 84

340962 Coding TGCATGCTGCTTAGTGCATC 85

340963 Coding ACTCCTGCATGCTGCTTAGT 86

340964 Coding ATCAGACTCCTGCATGCTGC 87

340965 Coding GCTATATCAGACTCCTGCAT 88

340966 Coding CCACAGCTATATCAGACTCC 89

340967 Coding GGCCACCACAGCTATATCAG 90

340968 Coding CTGCTGGCCACCACAGCTAT 91

340969 Coding AGCCCCTGCTGGCCACCACA 92

340970 Coding CATCCAGCCCCTGCTGGCCA 93

340971 Coding TTGTCCATCCAGCCCCTGCT 94

340972 Coding ATTGTCCATCCAGCCCCTGC 95

340973 Coding AGCGATTGTCCATCCAGCCC 96

340974 Coding TTTGAAGCGATTGTCCATCC 97

340975 Coding AGGGATTTGAAGCGATTGTC 98

340976 Coding CTTTCAGGGATTTGAAGCGA 99

340977 Coding GTAGCCTTTCAGGGATTTGA 100

340978 Coding CTCCAGTAGCCTTTCAGGGA 101

340979 Coding ACTTGCTCCAGTAGCCTTTC 102

340980 Coding AGTGAACTTGCTCCAGTAGC 103

340981 Coding TTATCAGTGAACTTGCTCCA 104

340983 Coding GCCAGTGAACTTATCAGTGA 105

340984 Coding CAGAGGCCAGTGAACTTATC 106

340985 Coding ACTCCCAGAGGCCAGTGAAC 107

340986 Coding GCCAGACTCCCAGAGGCCAG 108

340989 Coding TAGTTGGTCCTCAGGGCCAG 109

340990 Coding GTTGTTAGTTGGTCCTCAGG 110

340992 Coding AAGAGTTGGTGTTGTTAGTT 111

340993 Coding GGCTCAAGAGTTGGTGTTGT 112

340994 Stop Codon TCACGGCTCAAGAGTTGGTG 113

353939 Coding GGAGGCTTGTTCCATGTAGC 114

353947 Coding TCAGGGATTTGAAGCGATTG 115

353948 Coding CAGTAGCCTTTCAGGGATTT 116 # REGION SEQUENCE

353965 Stop Codon ATGGAGGTCTCACGGCTCAA 117

353971 3' UTR TAGATGGCCAGACACATCTG 118

353989 3' UTR TTGGGACAGCATGTTTAGGT 119

In one embodiment, the antisense inhibitors listed above are chimeric oligonucleotides ("gapmers") 20 nucleotides in length, composed of a central "gap" region consisting of eight 2'-deoxynucleotides, which is flanked on both sides (5' and 3' directions) by 3-nucleotide "wings." In this embodiment, the wings are composed of 2 '-0-(2-methoxyethyl) nucleotides, also known as (2'-MOE) nucleotides. In this embodiment, the internucleoside (backbone) linkages may be phosphorothioate (P=S) throughout the oligonucleotide. In this

embodiment, all cytidine residues may be 5-methylcytidines.

In one specific embodiment, the apoCIII inhibitor comprises or consists of 353982 (referred to below as AS02), GAGAATATACTTTCCCCTTA (SEQ ID NO: 50).

In embodiments where the subject is a human subject, the antisense apoCIII inhibitors may comprise an oligonucleotide that is less than 100% complementary to the human apoCIII mRNA (i.e.: having 1, 2, 3, 4, or 5 residues that are not complementary out of between 12-25 nucleotides of the inhibitor), which can result in the partial inhibition taken advantage of in the methods disclosed herein.

The inhibitor may be administered by any suitable route, including but not limited to oral, topical, parenteral, intranasal, pulmonary, or rectal in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. The term parenteral as used herein includes percutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, or intrathecal injection or infusion techniques and the like. In addition, there is provided a pharmaceutical formulation comprising an inhibitor disclosed herein and a pharmaceutically acceptable carrier. The inhibitor may be present in association with one or more non-toxic pharmaceutically acceptable carriers and/or diluents and/or adjuvants, and if desired other active ingredients. The inhibitor may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs.

In another embodiment, the inhibitor can also be administered locally via implantation of a membrane, sponge, or other appropriate material onto which the inhibitor has been absorbed or encapsulated, and delivery of the desired molecule can be via diffusion, timed- release bolus, nano-containers or continuous administration.

The dosage range depends on the choice of inhibitor, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner. For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, as is well understood in the art

Acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed. The therapeutic composition can contain formulation materials for modifying, maintaining, or preserving, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition. Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobials, antioxidants (such as ascorbic acid, sodium sulfite, or sodium hydrogen-sulfite), buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates, or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrins), proteins (such as serum albumin, gelatin, or immunoglobulins), coloring, flavoring and diluting agents, emulsifying agents, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (such as sodium), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (such as glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as pluronics; PEG; sorbitan esters; polysorbates such as polysorbate 20 or polysorbate 80; triton; tromethamine; lecithin; cholesterol or tyloxapal), stability enhancing agents (such as sucrose or sorbitol), tonicity enhancing agents (such as alkali metal halides - preferably sodium or potassium chloride - or mannitol sorbitol), delivery vehicles, diluents, excipients and/or pharmaceutical adjuvants (see, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES (18th Ed., A.R. Gennaro, ed., Mack Publishing Company 1990), and subsequent editions of the same, incorporated herein by reference for any purpose). The therapeutic compositions will be determined by a skilled artisan depending upon, for example, the intended route of administration, delivery format, and desired dosage. Such compositions can influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the inhibitor.

In a second aspect is provided methods for identifying a compound for treating or limiting development of obesity and/or fatty liver disease, comprising

(a) treating a first test animal with a high fat diet;

(b) treating a second test animal with (i) the high fat diet, (ii) a test compound; and (c) determining plasma apoCIII levels in the first test animal and the second test animal;

wherein test compounds that reduce plasma apoCIII levels and/or activity in the second test animal compared to plasma apoCIII levels and/or activity in the first test animal are candidate compounds for treating or limiting development of obesity and/or fatty liver disease.

The inventors have surprisingly discovered that reducing apoCIII expression and/or activity to control levels can both limit and reverse the deleterious effects of diet in obese subjects, subjects at risk of obesity, subjects with fatty liver disease, or subjects at risk of fatty liver disease, thus reducing body weight, reducing the rate of body weight increase in the obese subject or subject at risk of obesity, and treating or limiting development of fatty liver disease. Targeting apoCIII may thus be central to avoiding the negative consequences of an unhealthy diet, such as diabesity and/or fatty liver disease, and the methods can be used to identify therapeutics for treating or limiting development of obesity and/or fatty liver disease.

In one embodiment, reducing plasma apoCIII levels and/or activity in the second test animal compared to plasma apoCIII levels in the first test animal means to reduce apoCIII expression and/or activity in the second test animal between 10%-90% compared to apoCIII expression and/or activity in the first test animal. In various further embodiments, reducing apoCIII expression and/or activity in the second test animal means to reduce apoCIII expression and/or activity in the obese subject between about 10%-80%, 10%-70%, 10%- 60%, 10%-50%, 10%-40%, 10%-30%, 10%-20%, 20%-90%, 20%-80%, 20%-70%, 20%- 60%, 20%-50%, 20%-40%, 20%-30%, 30%-90%, 30%-80%, 30%-70%, 30%-60%, 30%- 50%, 30%-40%, 40%-90%, 40%-80%, 40%-70%, 40%-60%, 40%-50%, 50%-90%, 50%- 80%, 50%-70%, 50%-60%, 60%-90%, 60%-80%, 60%-70%, 70%-90%, 70%-80%, 80%- 90%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% compared to apoCIII expression and/or activity in the first test animal. Reducing plasma apoCIII levels and/or activity in the second test animal compared to plasma apoCIII levels in the first test animal means to reduce apoCIII expression and/or activity in the second test animal between about 30% and about 80% compared to apoCIII expression and/or activity in the first test animal

The test animals may be any suitable animal model of obesity, obesity risk, fatty liver disease, or fatty liver disease risk including dogs, cats, guinea pigs, rabbits, rats, and mice models of obesity, obesity risk, fatty liver disease, or fatty liver disease risk. In another embodiment, the animal model is a model of diabetic obesity or obesity risk.

In one embodiment, the method further comprises

(d) treating a third test animal with a non-high fat diet and determining plasma apoCIII levels and/or activity in the third test animal,

wherein test compounds that lead to plasma apoCIII levels and/or activity in the second test animal similar to plasma apoCIII levels and/or activity in the third test animal are candidate compounds for treating or limiting development of obesity or fatty liver disease.

The first and second test animals may be on any suitable high fat diet. In one embodiment, the high fat diet comprises on a caloric basis between about 40% and about 80% fat, about 10% to about 30% carbohydrate, and about 10% to about 30% protein. In another embodiment, the high fat diet comprises on a caloric basis about 60% fat, about 20% carbohydrate, and about 20% protein.

The third test animal may be on any suitable non-high fat diet. In one embodiment, the non-high fat diet comprises, on a caloric basis, about 5% to about 15% fat, about 50% to about 75% carbohydrates, and about 15% to about 40% protein. In another embodiment, the non-high fat diet comprises, on a caloric basis, about 11.4% fat, about 63% carbohydrates, and about 26% protein.

In another embodiment, the methods further comprise large-scale synthesis of the test compounds that reduce plasma apoCIII levels and/or activity in the second test animal compared to plasma apoCIII levels and/or activity in the first test animal.

The test compound(s) may be any suitable test compounds, including but not limited to nucleic acids, polypeptides, and small molecules. When the test compounds comprise polypeptide sequences, such polypeptides may be chemically synthesized or recombinantly expressed. Recombinant expression can be accomplished using standard methods in the art, as disclosed above. Such expression vectors can comprise bacterial or viral expression vectors, and such host cells can be prokaryotic or eukaryotic. Synthetic polypeptides, prepared using the well-known techniques of solid phase, liquid phase, or peptide

condensation techniques, or any combination thereof, can include natural and unnatural amino acids. Amino acids used for peptide synthesis may be standard Boc (Na-amino protected Na- t-butyloxycarbonyl) amino acid resin with standard deprotecting, neutralization, coupling and wash protocols, or standard base-labile Na-amino protected 9-fluorenylmethoxycarbonyl (Fmoc) amino acids. Both Fmoc and Boc Να-amino protected amino acids can be obtained from Sigma, Cambridge Research Biochemical, or other chemical companies familiar to those skilled in the art. In addition, the polypeptides can be synthesized with other Na-protecting groups that are familiar to those skilled in this art. Solid phase peptide synthesis may be accomplished by techniques familiar to those in the art and provided, such as by using automated synthesizers.

When the test compounds comprise antibodies, such antibodies can be polyclonal or monoclonal. The antibodies can be humanized, fully human, or murine forms of the antibodies. Such antibodies can be made by well-known methods, such as described in Harlow and Lane, Antibodies; A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1988).

When the test compounds comprise nucleic acid sequences, such nucleic acids may be chemically synthesized or recombinantly expressed as well. Recombinant expression techniques are well known to those in the art (See, for example, Sambrook, et al., 1989, supra). The nucleic acids may be DNA or RNA, and may be single stranded or double. Similarly, such nucleic acids can be chemically or enzymatically synthesized by manual or automated reactions, using standard techniques in the art. If synthesized chemically or by in vitro enzymatic synthesis, the nucleic acid may be purified prior to introduction into the cell. For example, the nucleic acids can be purified from a mixture by extraction with a solvent or resin, precipitation, electrophoresis, chromatography, or a combination thereof. Alternatively, the nucleic acids may be used with no or a minimum of purification to avoid losses due to sample processing.

When the test compounds comprise compounds other than polypeptides, antibodies, or nucleic acids, such compounds can be made by any of the variety of methods in the art for conducting organic chemical synthesis. All references cited are herein incorporated by reference in their entirety. Within this application, unless otherwise stated, the techniques utilized may be found in any of several well-known references such as: Molecular Cloning: A Laboratory Manual (Sambrook, et al.,

1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, CA),

"Guide to Protein Purification" in Methods in Enzymology (M.P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al.

1990. Academic Press, San Diego, CA), Culture of Animal Cells: A Manual of Basic Technique, 2nd Ed. (R.I. Freshney. 1987. Liss, Inc. New York, NY), Gene Transfer and Expression Protocols, pp. 109-128, ed. E.J. Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin, TX).

As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. "And" as used herein is interchangeably used with "or" unless expressly stated otherwise.

As used herein, "about" means +/- 5% of the recited value.

All embodiments of any aspect disclosed herein can be used in combination, unless the context clearly dictates otherwise.

Unless the context clearly requires otherwise, throughout the description and the claims, the words 'comprise', 'comprising', and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to". Words using the singular or plural number also include the plural and singular number, respectively. Additionally, the words "herein," "above," and "below" and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of the application.

The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While the specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize.

Examples:

A major cause of the increasing number of subjects with obesity and type-2 diabetes (T2D) is the imbalance between energy intake and expenditure and the failure of β-cells to compensate for the need of increased insulin secretion to maintain glucose homeostasis. The initial phase in the progression to T2D is characterized by peripheral tissue insulin resistance with a compensatory increase in secretion of insulin by the β-cells. However, over time the β-cells start to fail and T2D occurs when there is an imbalance between the secreted insulin and the increased need, caused by the resistance in the peripheral insulin target tissues.

Apolipoprotein CIII (apoCIII) is an 8.8 kDa polypeptide mainly synthesized in the liver. In the current study we have examined whether apoCIII can participate in the progression from insulin resistance to impaired glucose tolerance (IGT) and overt T2D upon exposure to high-fat diet (HFD). Further, we aimed to clarify if lowering the apolipoprotein with an antisense oligonucleotide (ASO) directly targeting the apoCIII gene can be a successful therapeutic tool to reverse and/or prevent HFD-induced obesity and T2D in mice. To investigate these questions, we used three different experimental set ups (Figs. 6a, 7a, 11a) of 8-week old male C57B16/j mice undergoing a HFD intervention and treated either with an inactive or scrambled (Scr) ASO (Scr+HFD) or with an active ASO (ASO+HFD) targeting apoCIII. Mice on chow diet were used as healthy Controls.

To examine whether apoCIII is a player in the progression from insulin resistance to impaired glucose tolerance and overt diabetes upon HFD exposure, four groups of C57BL/6J mice with different treatments were studied. The control group was fed a standard chow diet and the three remaining groups were all on HFD. Additionally, two of them were given i.p. injections with either antisense apo CIII, reducing the levels of the apolipoprotein to undetectable levels, or a scrambled (scr) inactive, not affecting the levels of the apolipoprotein (data not shown). ApoCIII in plasma was evaluated by Western blotting, as there is no reliable method for quantitative measurements. Treatments started when the mice were 8 weeks old. Body weight (BW) was measured once per week and after two months the first intraperitoneal glucose tolerance test (IPGTT) was performed. After this time period there was an equivalent increase in BW in all mice on HFD and only the control group had a normal glucose handling and, as expected, a lower weight. These results are in line with those showing that apoCIII knock-out mice, totally lacking apoCIII, are more sensitive to diet induced obesity and insulin resistance due to absence of inhibition of lipoprotein lipase (LPL) by the apolipoprotein and thereby enhanced uptake of fatty acids from triglycerides in adipose tissue. We decided on a modified treatment protocol. The group on only HFD was now instead treated with an apoCIII antisense that does not eliminate, but reduces the levels of the apolipoprotein to control levels.

Lowering apoCIII reverses obesity, insulin resistance and IGT

To discard potential effects of the inactive Scr ASO on the glucometabolic outcomes of the HFD, we monitored body weight (BW) in untreated mice on HFD and Scr+HFD animals for the first 10 weeks of study in protocol 1, and compared to control diet-fed mice. All animals on HFD significantly increased BW (Fig. la) and weight gain as compared to Controls independently of the treatment with the inactive Scr ASO. BW increase was already statistically higher after 2- week dietary intervention and progressively increased until week 10 of study in both Scr- treated and untreated HFD mice (Fig. la). Additionally, we performed an i.p glucose tolerance test (IPGTT) after 8 weeks of study and could confirm that both untreated and Scr- treated mice on HFD exhibited similar IGT as compared to Controls. These results validated the Scr ASO as an inactive treatment that could be used in our study as a vehicle since it did not affect the glucometabolic impact of the HFD.

To examine if lowering apoCIII could reverse diet- induced obesity (DIO), insulin resistance and IGT, we started to administer an active ASO against apoCIII to the untreated mice on HFD. The active ASO started to be i.p injected in this group of mice after 10 weeks of study, when they DIO and IGT were already stablished. We observed that the active ASO against apoCIII stopped weight gain in HFD-fed mice already after 3 weeks of treatment .

Weight loss was already statistically significant in ASO+HFD as compared to their Scr+HFD counterparts after 5 weeks of ASO treatment. Furthermore, ASO+HFD mice progressively reduced their BW and weight gain during the time course of the study reaching similar BW to those mice on chow diet at the end of the study (Fig. la). Reduction in BW and weight gain in ASO+HFD mice caused obvious differences in body configuration between ASO+HFD and Scr+HFD mice leading to a lean phenotype despite consumption of a HFD (Fig. lb).

Food intake was similar between ASO+HFD and Scr+HFD mice, but significantly lower when compared to Controls . When food intake was corrected by the energy supply of the diets (Chow diet, 2.99 kcal g^"1; HFD, 5.24 kcal g^"1) we showed that calorie intake was the same in all groups. Notably, HFD significantly increased feed efficiency in Scr+HFD animals, whereas the weight loss observed in the ASO+HFD mice reduced this metabolic parameter down to normal values. These results suggest that, whereas the obese phenotype of the Scr+HFD was due to more efficient energy storage in terms of fat, the weight loss observed in the ASO+HFD could be attributable to higher energy expenditure since it was independent on the fat overloading from the HFD.

The reversion of the obese phenotype together with the recovery of the energy efficiency in HFD mice treated with the active ASO were paralleled by a significant reduction of the circulating apoCIII as compared to Scr+HFD animals; the later exhibiting a large increase of the circulating levels of the apolipoprotein as compared to Controls (Fig. lc).

We next investigated whether lowering apoCIII with ASO in HFD mice could improve insulin sensitivity, i.p insulin tolerance test (IPITT) performed at the end of the study showed that ASO+HFD mice exhibited significantly improved insulin sensitivity as compared to the insulin resistant Scr+HFD mice (Fig. Id). Moreover, lowering apoCIII with ASO treatment steadily improved the glucometabolic phenotype of the ASO+H D mice (Fig. 6g-i, Fig. le). IPGTT monitoring showed that, ASO+HFD mice exhibited lower blood glucose excursion during IPGTT as compared to Scr+HFD after 6 weeks of ASO treatment (16 weeks of study). From this time point until the end of the study, ASO+HFD mice showed a significantly better performance in the IPGTT as compared to Scr+HFD ones (Fig. le).

Several known mechanisms underlie the development and progression of insulin resistance such as glucose toxicity, lipid-mediated insulin resistance and ectopic fat accumulation. Lipid profiles determined by size-exclusion chromatography showed that triglycerides (Tgs) associated to very low-density lipoproteins (VLDL-Tgs) and

intermediate/low-density lipoproteins (IDL/LDL-Tgs), but not high-density lipoproteins

(HDL-Tgs), as well as total Tgs, were significantly lowered in ASO+HFD mice as compared to their Scr-HFD counterparts (Fig. If,). Similarly, all plasma eluted lipoprotein particles associated to cholesterol, as well as total cholesterol (Choi), were significantly reduced in ASO-treated mice on HFD as compared to Scr+HFD ones (Fig. lg). Furthermore, the lipotoxic effects of the HFD deteriorating liver morphology in the Scr-treated mice were completely reversed by lowering apoCIII with ASO treatment in HFD-fed animals (Fig. lh). These changes were attributable to a significant reduction in the hepatic lipid accumulation in ASO+HFD as compared to Scr+HFD mice, exhibiting these last ones a pronounced liver steatosis (Fig. li).

The drastic effect of the ASO treatment against apoCIII reversing obesity, insulin resistance and IGT raises the possibility that apoCIII might play a causative role in the progression from diet-induced insulin resistance to IGT and, later, T2D. Early effects of ASO treatment reversing insulin resistance and IGT

We next investigated if ASO treatment lowering apoCIII is able per se to initiate the reversion of insulin resistance and IGT, independently on the reduction of the body weight. To do that, we used an experimental set up where animals were subjected to a HFD

intervention during 10 weeks and, thereafter, treated either with the Scr or with the active ASO against apoCIII during 4 additional weeks. This was based on the knowledge that the active ASO against apoCIII did not induce a significant reduction in BW or weight gain until week 5 of treatment in protocol 1 (Fig. la).

During the first 10 weeks of study, animals on HFD significantly increased BW and weight gain as compared to Controls (Fig. 2a). As shown before, ASO+HFD mice stopped gaining weight after 3 weeks of treatment, but ASO treatment did not induce any significant reduction in BW or weight gain as compared to Scr- treated mice (Fig. 2a). In good agreement with these results, NMR studies confirmed that ASO+H D and Scr+HFD mice presented a similar body configuration in terms of fat and lean mass (Fig. 2b,c). However, the active ASO efficiently reduced circulating apoCIII in ASO+HFD mice as compared to Scr+HFD animals; the later exhibiting a significant increase of the circulating levels of the apolipoprotein as compared to Controls (Fig. 2d).

Monitoring of IPITT and IPGTT showed that during the first 10 weeks of study, HFD mice exhibited insulin resistance and IGT (not shown). We could observe that 2-week on ASO treatment was not enough to improve IPITT and IPGTT in ASO+HFD mice as compared to Scr- treated ones on HFD. Interestingly, mice treated for 4 weeks with the active ASO against apoCIII showed an improved performance in the IPITT (Fig. 2e) and in the IPGTT (Fig. 2f) as compared to Scr+HFD mice. Of importance is to note that these improvements occurred when ASO+HFD mice were still obese and debuted in the absence of recovery in any of the parameters determined in the metabolic cages (Fig. 2g-j). These results suggest that the reduction of apoCIII in HFD-fed mice might per se be a potential cause impeding the progression from lipid-mediated insulin resistance to IGT and, further, T2D.

In fact, we could observe that HFD significantly decreased body temperature during the first 10 weeks of study and that active ASO lowering apoCIII induced a partial recovery in this parameter after 4 weeks of treatment . As body temperature is intimately related to the ability of the brown adipose tissue (BAT) to burn fatty acids for heat production, we investigated a number of genes involved in brown fat thermo genesis.. We found that only the peroxisome proliferator- activated receptor-gamma coactivator 1 alpha (Pgcla) was significantly upregulated in the BAT of the ASO+HFD mice as compared to the Scr-treated ones on HFD; although an increasing tendency was observed in the Uncoupling protein 1 (Ucpl) and in the peroxisome proliferator-activated receptor alpha (Ppara) gene expression levels in ASO-treated mice on HFD as compared to the Scr+HFD ones. Pgcla upregulation in BAT can be explained by the improved insulin sensitivity displayed by ASO+HFD mice, but might not be sufficient alone to fully explain the increased body temperature in these animals. Nevertheless, activation of BAT has been shown to increase the clearance of plasma triglycerides, a process crucially dependent on local lipase activity and fatty acid translocase CD36. Here, we found that the gene expression of the transmembrane receptor Cd36 was upregulated in ASO+HFD as compared to Scr+HFD mice. We also observed a slight, but still significant, upregulation in the mRNA levels of the Atgl. Based on this, it can be deduced that receptor- mediated uptake of lipids probably plays a major role in BAT activation secondary to improved insulin sensitivity upon lowering apoCIII, thus increasing fatty acid catabolism for heat production.

Early effects of ASO treatment reversing dyslipidemias and hepatic steatosis

We examined plasma lipid profiles after 4 weeks of ASO treatment and could observe that active ASO lowered VLDL-Tgs, but not HDL-Tgs, reaching statistical significance for IDL/LDL-Tgs and total Tgs as compared to Scr+HFD mice (Fig. 3a). Similarly, a tendency towards lowering all plasma eluted lipoprotein particles associated to cholesterol was detected in ASO+HFD mice, being total cholesterol significantly lower in this group as compared to their Scr+HFD counterparts (Fig. 3b).

These results led us to examine other peripheral organs involved in lipid metabolism that could contribute to take up circulating lipids for further utilization and/or storage. We then performed gene profiling in visceral and subcutaneous adipose tissues. qPCR

experiments in visceral adipose tissue (VAT) showed a robust increase in Ucpl gene expression together with a significant upregulation of Pgcla, tumor necrosis factor receptor superfamily member 9 (Cell 37) and adipose triglyceride lipase (Atgl) in ASO+HFD mice as compared to Scr+HFD animals. However, the rest of the genes examined remained unchanged between Scr- and ASO-treated groups and were downregulated in both of them as compared to mice on chow diet. These results indicate there are early phenotypical changes improving thermogenesis and lipid utilization in the VAT already after 4-week ASO treatment, despite consumption of a HFD. We did not observe significant changes in genes involved in thermogenesis, beige/brite processes and/or lipid uptake and utilization in the SAT of ASO+HFD after 4- week treatment as compared to Scr+HFD, excepting for Cell 37 which was significantly upregulated in the SAT from ASO+H D mice. However, all other genes involved in the biochemical pathways we analyzed in the SAT remained downregulated in both Scr- and ASO-treated mice on HFD as compared to Controls.

Obtained results prompted us to investigate early changes induced by ASO treatment in the main source for apoCIII production and secretion, the liver. Oil Red Ό' (ORO) staining in liver sections revealed that Scr-treated mice on HFD presented hepatic steatosis and that ASO treatment targeting apoCIII considerably reduced the amount of lipids visualized in liver sections from ASO-HFD mice; although both groups exhibited substantially higher hepatic lipids as compared to Controls (Fig. 3c). These results were confirmed spectrophotometrically by measuring liver Tgs content (Fig. 3d). Hepatic steatosis in Scr+HFD mice was

accompanied by an upregulation in their liver apoCIII gene expression, which was repressed by ASO treatment (Fig. 3e). The robust reduction in hepatic apoCIII mRNA levels showed by ASO+HFD mice was paralleled by a significant upregulation in hepatic lipase (Lipc) gene expression, being this repressed in Scr-treated mice due to their higher liver apoCIII mRNA levels (Fig. 3f). The reduction of liver and circulating apoCIII due to ASO treatment might be responsible for an enhanced hepatic lipid catabolism and for an increased liver-mediated lipoprotein clearance. These aspects were confirmed when we examine target genes involved in hepatic uptake of lipoproteins (Fig. 3g-i) as well as pathways promoting liver lipid catabolism (Fig. 3j-l). Increased lipoprotein uptake is mediated by the upregulation in hepatic LDL-receptor (Lldr) (Fig. 3g) and Cd36 (Fig. 3h), but not in scavenger receptor class B member 1 (Srbl) (Fig. 3i) in ASO+HFD mice as compared to Scr-treated ones in HFD, thus explaining the improvements in the systemic lipid profiles observed after 4-week ASO treatment. The reduction of plasma lipoproteins upon lowering apoCIII might also trigger lipolytic pathways via Ppara activation, which in turn activates fatty acids β-oxidation in the liver. In good agreement, we could find that 4- week ASO treatment significantly upregulated hepatic Ppara (Fig. 3j) and the mitochondrial fatty acid β-oxidation gene carnitine

palmitoiltransferase I (CptT) (Fig. 3k), but not retinoid X receptor (Rxr) (Fig. 31), as compared to Scr-treated mice.

Last, we investigated whether the reduction in cholesterol lipoprotein profile observed in HFD mice administered for 4 weeks with the active ASO against apoCIII could be related to a gain-of-function of genes related to cholesterol efflux from liver into the bile acids. We, therefore, examine early changes in gene expression of hepatic adenosine triphosphate- binding cassette transporter G5/8 (Abcg5/Abcg8) and found that both genes were significantly upregulated in ASO+HFD as compared to Scr+HFD mice (Fig. 3m). These changes might account for an increased hepatobiliary cholesterol efflux, thus reducing plasma lipoprotein particles associated to cholesterol.

These entire features can potentially represent the molecular origin by which lowering apoCIII improves systemic lipid profiles and reduces hepatic fat accumulation, thus reversing insulin resistance and IGT. Furthermore, these findings open new therapeutic avenues not only for treatment but also for prevention of lipid-mediated insulin resistance, IGT and, further, T2D, by targeting apoCIII with ASO.

Lowering apoCIII prevents obesity, insulin resistance and IGT

To further investigate whether the active ASO lowering apoCIII could prevent the harmful effects of the HFD, we used an experimental set up where mice started a HFD intervention being simultaneously treated with either the inactive Scr or with the active ASO against apoCIII . We observed that Scr+HFD and ASO+HFD groups significantly increased BW and weight gain during the first 3 weeks of study (Fig. 4). ASO+HFD mice showed a slight (non- significant) reduction in BW after 4- week treatment as compared to Scr+HFD ones, which reached statistical significance after 5-week of ASO administration (Fig. 4a). From this time point until the end of the study, ASO+HFD stopped gaining weight and their BW evolution during the time course of the study was similar to those mice on chow diet (Fig. 4a). NMR studies showed that the differences in body configuration between ASO+HFD and Scr+HFD mice were due to a significant reduction in fat mass, without changes in the lean mass (Fig. 4b,c). These changes were paralleled by a significant reduction of the circulating apoCIII in the ASO+HFD mice as compared to Scr+HFD animals; the later exhibiting a large increase of the circulating levels of the apolipoprotein as compared to Controls (not shown).

Monitoring of IPITT showed that ASO treatment against apoCIII counteracted the development of insulin resistance as the blood glucose excursion of the ASO+HFD mice was comparable to the control group and was significantly lower to what observed in the insulin- resistant Scr-HFD animals . These changes were already detectable after 4 weeks of study and persisted during the whole time course of the study until the end. IPGTT was slightly, but not significantly, improved after 4 weeks of study in

ASO+HFD as compared to Scr+HFD mice. Interestingly, IPGTT progressed towards an improved glycemic control in ASO+HFD animals along the study compared to Scr+HFD mice ; and by the end of the study ASO-treatment completely re-established glucose homeostasis as shown by a normoglycemic phenotype comparable to that observed in the IPGTT of the mice on chow diet.

ASO-treated mice on HFD exhibited increased 0₂ consumption, CO2 production and, consequently, a higher RER as compared to Scr+HFD animals. These changes, all indicative of higher fuel utilization in ASO+HFD mice, were not attributable to changes in food intake, calorie intake or physical activity, but to higher energy expenditure. Moreover, we could show that the lower temperature detected in the Scr+HFD mice as compared to Controls was completely re-established up to normal levels upon ASO administration in all time points where this parameter was evaluated in our study. These results indicate that ASO treatment could induce phenotypical changes in the BAT promoting the activation of thermogenic pathways, as well as genes involved in lipid uptake and metabolism. We could observe that ASO treatment prevented the obvious morphological deteriorations showed in the BAT of Scr+HFD mice. The improvements in BAT morphology detected in ASO+HFD mice were accompanied by upregulations of the expression levels of genes activating thermogenic pathways, such as Ucpl, Ppara, Ppary, Pgcla and cell death activator (Cidea), but not PR domain containing 16 (Prdml6). Moreover, ASO+HFD mice displayed an upregulation in the gene expression of Cd36 and Atgl (. Of importance is to note that genes involved in all biochemical pathways we analyzed in the BAT were downregulated in Scr+HFD as compared to Controls. Altogether these results suggest that lowering apoCIII with ASO treatment increases fatty acid utilization for heat production in the BAT and that this activation of the BAT could also contribute to a higher clearance of circulating lipids in ASO- treated mice on HFD.

Lowering apoCIII prevents dyslipidemias and NAFLD

We could observe that ASO+HFD mice showed significantly reduced adipocyte size in VATand SAT sections as compared to Scr+HFD, being the area of the visceral and the subcutaneous adipocytes of the ASO-treated mice comparable to those from chow diet-fed mice. Gene expression data showed a robust upregulation (~ 10-fold) in Ucpl expression levels in VAT from ASO+H D as compared to Scr-HFD mice (. These changes were accompanied by an increase in other thermogenic genes as well as beige/brite markers, such as Pgcla, Prdml6, Cdl37 and Tmem26. Furthermore, gene expression analysis in SAT from ASO+HFD mice revealed a significant upregulation in all the genes involved in the biochemical pathways studied in the current work. Altogether, these results evidence that the preventing effects of the ASO treatment on the glucometabolic phenotype observed in the ASO+HFD animals might be also explained by a higher activation of the thermogenic pathways in white adipose tissues (WAT). These features, together with the increased beige/brite markers and the upregulation of lipid uptake and metabolism in WAT from ASO+HFD, confirm an active role of the WAT dissipating the excess of energy from the HFD, a process classically assigned to the BAT.

However, as the liver is known to be a main producer of apoCIII in the body and, hence, a main target tissue for the active ASO, we hypothesized that the molecular mechanism by which lowering apoCIII with ASO treatment leads to a normolipidemic, lean and insulin sensitive phenotype, resides in the liver. Staining with ORO revealed that the NAFLD observed in the Scr+HFD mice was prevented by lowering apoCIII with active ASO (Fig. 5c). These results were confirmed by measuring liver Tgs content by spectrophotometry (Fig. 5d). Gene expression experiments in the liver showed that ASO treatment induced a robust reduction (~6-fold) in hepatic apoCIII mRNA levels, as compared to Scr-treated mice on HFD (Fig. 5e). The significant increase in apoCIII expression levels upon HFD feeding observed in Scr+HFD animals was accompanied by a drastic reduction in the expression levels of Lipc, which was recovered up to almost normal levels in ASO+HFD mice (Fig. 5f). The upregulation of Lipc in ASO+HFD animals indicates that the Tgs-lowering action of the ASO against apoCIII can be attributable to enhanced lipolytic pathways in the liver, thus explaining the reduction of hepatic lipid accumulation down to normal levels in ASO-treated mice despite consumption of a HFD. Furthermore, it has been shown that Lipc also exert functions as ligand/bridging factor for receptor-mediated uptake of lipoproteins. In line with this, we found that ASO treatment significantly upregulated genes involved in lipoproteins uptake, such as Ldlr (Fig. 5g), Srbl (Fig. 5h) and the fatty acid translocase Cd36 (Fig. 5i), which might be responsible for the improved systemic lipid profiles observed in the

ASO+HFD mice as compared to their Scr+HFD counterparts, we also found that ASO+HFD mice exhibited an upregulation of liver Ppara (Fig. 5j), Rxr (Fig. 5k) and the mitochondrial fatty acid β-oxidation gene Cptl (Fig. 51), as compared to their Scr+HFD counterparts. Finally, we could observe that ASO+HFD mice displayed a gain-of-function in the hepatic Abcg5 and Abcg8 genes as compared to the Scr-treated ones on HFD. These results confirmed that lowering apoCIII induces hepatobiliary cholesterol efflux, thus contributing for the reduction in plasma lipoprotein particles associated to cholesterol.

In summary, we have identified a liver-dependent mechanism by which lowering apoCIII with ASO treatment is able to reverse and prevent diet-induced obesity and the progression from insulin resistance to IGT and hence, later, T2D. We have shown that the molecular origin accounting for the reversion of these diseases upon apoCIII reduction is triggered by impeding lipid-mediated insulin resistance, and that this is due to the activation of the hepatic catabolic and excretion machineries for triglycerides and cholesterol, respectively. Importantly, we confirmed that same mechanisms are able to prevent obesity and T2D when mice were simultaneously given a HFD and treated with an active ASO targeting apoCIII.

Our findings also challenge the assumption that weight-loss can be the triggering factor for the recovery of insulin resistance and IGT observed in our mouse models. Our results showed that early improvements on insulin sensitivity and glucose tolerance were attributable to the lowering of apoCIII per se, since they preceded weight loss and debuted prior to the recovery in any of the parameters related to energy metabolism. However, we cannot discard that weight-loss and reduced adiposity might play a positive secondary role when preventing lipid-induced insulin resistance and IGT with ASO treatment by promoting the thermogenic actions of BAT and WAT. In this scenario, we have shown that BAT and WAT can act as metabolic regulators of Tgs-rich lipoproteins (TRL) uptake from circulation for further utilization of the cleared Tgs as source of fuel. In conditions of reduced apoCIII, fatty acids can be efficiently channeled into BAT and WAT due to a metabolic program that boosts TRL uptake and its catabolism. The uptake process is associated with increased permeability for lipoproteins and is crucially dependent on local lipases and the fatty acid translocase CD36; whereas the lipid utilization in BAT and WAT is fundamentally mediated by the activation of thermogenic pathways. As these

improvements seem to be more directly related to weight-loss and reduced adiposity, rather than to the lowering of apoCIII by itself, we propose that the mechanism by which ASO treatment results in a normolipidemic, normoglycemic and insulin sensitive phenotype, despite consumption of a HFD, resides in the liver. In conclusion, our findings reveal how imperative it is to understand the full extent of apoCIII functions in the context of lipid-mediated insulin resistance to provide new therapeutic approaches focus in lowering apoCIII for the treatment and prevention of diet- induced obesity and T2D.

Methods

Animals.

C57B16/j male mice were purchased from Charles River, USA. Age-matched mice were used in all studies. All animals were acclimated to our animal facilities for a period of 2 weeks prior starting the experiments and had ad libitum access to chow and water. Mice were housed 3-6 animals per cage in a temperature- and humidity-controlled room with 12 h light- 12 h dark cycles. Animal care and experimentations were carried out according to the Animal Experiment Ethics Committee at Karolinska Institutet, Stockholm, Sweden.

Diets.

At 8 weeks of age, weight-matched mice were randomly divided into two groups with a similar average of body weight and assigned either to standard chow (R70, Lantmannen, Sweden) or HFD (Open Source Diets D 12492, Research Diets, New Brunswick, NJ, USA). On calorie basis, the normal chow provided 2.99 kcal g^"1 (% of kcal from: fat 11.4%; proteins 25.8%; and carbohydrates 62.8%) and the HFD provided 5.24 kcal g^"1 (% of kcal from: fat 60%; proteins 20%; and carbohydrates 20%).

Antisense Oligonucleotides.

Antisense oligonucleotides were provided by Ionis Pharmaceuticals, Inc. (Carlsbad, USA). Sequences of the antisense oligonucleotides were as follows: Active antisense (ASO) against apoCIII gene (ION-353982, 5 ' -GAGAATATACTTTCCCCTTA-3 ' SEQ ID NO: 50); and inactive or scrambled (Scr) antisense (ION- 141923 , 5 '-CCTTCCCTGAAGGTTCCTCC- 3' SEQ ID NO: 120), used as a control. Underlined sequences indicate the 2'-0-methoxyethyl- phosphorothioate modified bases. Efficacy and security profiles were ensured by Ionis Pharmaceuticals, Inc. (Carlsbad, USA).

Experimental design.

Three experimental set ups were used in the current study, namely protocol 1, protocol

2 and protocol 3. Mice in protocol 1 were assigned either to HFD or to standard chow. Diet intervention started at the age of 8 weeks and lasted for 24 additional weeks. At start, mice assigned to the HFD group were divided into two: One receiving a weekly i.p injection of the Scr antisense (12.5 mg kg ) and the other one remained untreated. HFD-fed mice were subjected to the above mentioned procedure during the first 10 weeks of the study to ensure that the Scr antisense did not affect the obesogenic/diabeto genie properties of the HFD. After 10 weeks on diet, the untreated HFD group started to receive two i.p injections per week of the active ASO (25 mg kg^"1) during the last 14 weeks of the study. Animals fed with chow diet during 24 weeks did not receive any antisense treatment and were used as healthy controls. Mice in protocol 2 were assigned either to HFD or to standard chow. Diet intervention started at the age of 8 weeks and lasted for 14 additional weeks. After 10 weeks on diet, HFD-fed mice were divided into two groups: One receiving two i.p injections per week of the Scr antisense (25 mg kg^"1) and the other one administered twice per week with the active ASO (25 mg kg^"1) during the last 4 weeks of the study, respectively. Animals fed with chow diet during 14 weeks were given two i.p injections of saline during the whole time course of the study and were used as healthy controls. Mice in protocol 3 were assigned either to HFD or to standard chow. Diet intervention started at the age of 8 weeks and lasted for 14 additional weeks. At start, mice assigned to the HFD group were divided into two: One receiving two i.p injections per week of the Scr antisense (25 mg kg^"1) and the other one administered twice per week with the active ASO (25 mg kg^"1) during the whole time course of the study. Animals fed with chow diet during 14 weeks were given two i.p injections of saline during the whole time course of the study and were used as healthy controls. All groups in each protocol will be referred to as Control, Scr+HFD and ASO+HFD, respectively. The protocol to which each experimental group and their controls belong will be specified in each case and where it corresponds. Body weight was monitored twice per week in all

experimental set ups used in the current study.

IPITTs and IPGTTs.

IPITTs were performed after 12-h overnight starvation for animals in protocol 1 and after 6-h fasting during day time for animals in protocols 2 and 3. In protocol 1, IPITT was performed at the end of the experiment. In protocols 2 and 3, IPITTs were performed after 4, 8, 10, 12 and 14 weeks of study. In the IPITTs, fasting glucose (0 time point) was measured followed by an i.p injection of insulin (0.25 U kg^"1 of body weight). Subsequently, blood glucose was measured again 10 min later and, immediately after, mice received an i.p injection of glucose (1 g kg^"1 of body weight), as previously described³¹. Blood glucose levels were measured at 15, 30, 60, 90 and 120 min after glucose administration using a FreeStyle Lite™ glucose meter (Abbot Diabetes Care). IPGTTs were performed after 12-h overnight starvation for animals in protocol 1 and after 6-h fasting during day time for animals in protocols 2 and 3. In protocol 1, IPGTTs were performed after 8 weeks of study and every 4 weeks until the end of the experiment. In protocols 2 and 3, IPGTTs were performed after 4, 8, 10, 12 and 14 weeks of study. In the IPGTTs, basal blood glucose (0 time point) was measured in samples drawn from the tail vein. Thereafter, mice received an i.p injection of glucose (1.5 g kg^"1 body weight), and glucose levels were measured after 10, 30, 60 and 120 min using a FreeStyle Lite glucose meter (Abbot Diabetes Care).

Insulin secretion during IPGTTs.

Insulin levels were determined in plasma samples from blood collected from the tail vein at the same time points as for the IPGTT in animals from protocols 2 and 3. In vivo glucose-stimulated insulin secretion was analyzed in plasma samples using an ultrasensitive mouse insulin enzyme-linked immunosorbent assay (ELISA) Kit (Crystal Chem Inc, Downers Grove, IL).

Size-exclusion chromatography (SEC) analysis for plasma lipid profiles.

Lipid profiles were obtained by size-exclusion chromatography. Plasma samples from three subsets of non-fasting mice on HFD following the same Scr or ASO treatment procedures were used for these experiments. Plasma lipoproteins were separated by size class using a LaChrom Elite™ HPLC system (Hitachi, Germany) and Superose™ 6 PC 3.2/300 gel column (GE Helthcare, Sweden). Plasma triglycerides and cholesterol concentrations were determined using GPO-PAP and CHOD-PAP methods (Roche, Switzerland), respectively. The triglycerides and cholesterol associated to the different lipoprotein fractions were measured as the area under the curve using EZChrome™ Elite software (Aglient

Technologies, Germany).

Food and calorie intake measurements.

In animals belonging to protocol 1, food intake of Control, Scr+HFD and ASO+HFD, was measured for the last 5 consecutive weeks of the study. All animals and the amount of food in the cage were weighted twice a week. Food intake was calculated by dividing grams of food consumed per day per number of animals in the cage. Calorie intake was calculated by multiplying the food intake per either 2.99 kcal g^"1 in the case of the chow diet or 5.24 kcal g^"1 in the case of the HFD. Finally, calorie efficiency, presented as body weight gain per kcal consumed per mouse, was determined to establish the relationship between body weight gain and amount of calories consumed by each animal. The average of all weeks for every parameter was presented in the figures.

Energy metabolism characterization in the metabolic cages.

Two subsets of mice on HFD for 14 weeks, starting at 8 weeks of age, were treated either with the Scr antisense or with the active ASO following the same treatment procedures. At the end of the studies, all mice were acclimated for 24 h in single cages. Subsequently, they were monitored for 4 consecutive days in the Oxymax™ Lab Animal Monitoring System (Comprehensive Laboratory Animal Monitoring System, CLAMS; Columbus Instruments, Columbus, OH) with ad libitum access to food except for a 12-h fasting period during the last night. The following parameters were continuously monitored: 0₂ consumption (VO2), CO2 production (VCO2), respiratory exchange ratio, food intake, calorie intake, energy expenditure and movement. Movement was reported as total beam breaks for the XYZ axis. Energy expenditure was calculated as the relationship between heat and body weight of each individual animal, and presented in the figures as kcal per kg of mouse per h.

Body temperature.

Body temperature was monitored in animals belonging to protocol 2 and 3 after 4, 8, 10, 12 and 14 weeks of study using a FLUKE™ 5 1 K/J thermometer (Fluke Corporation). Body composition.

The same subsets of mice used for energy metabolism studies in the metabolic cages were also subjected to an EchoMRI-100™ system (Echo Medical Systems, USA) to assess total lean and fat mass. The measurement is based on nuclear magnetic resonance (NMR) that takes advantage of the difference in density of the hydrogen nuclei in adipose tissue, water and bone. These experiments were performed in ad libitum conditions and without anesthesia.

Histological studies.

Histological evaluation in livers from animals in protocol 1 was performed by H&E staining in tissue cryosections. Briefly, livers were dissected out, washed in PBS, frozen in liquid nitrogen and stored at -80°C until use. Tissues were sectioned into 20μιη thick sections with a cryostat (Microm™ HM500M/Cryostar NX70; Thermo Scientific) and collected onto SuperFrost Plus™ microscope slides (VWR International). After 2 h equilibrating at room temperature, liver sections were formalin-post-fixed during 30 min, stained with H&E (Histolab) and mounted VectaMount permanent mounting medium (Vector Laboratories, Inc.). Pictures were taken at 20X and 40X magnification objectives in an optical microscope (Leica).

For histological analysis of BAT, VAT and SAT from animals in protocols 2 and 3, H&E staining was performed in formalin-fixed tissues. For that purpose, mice were anesthetized with isoflurane and transcardially perfused with PBS followed by freshly prepared 4% paraformaldehyde in PBS. Adipose tissues were dissected out and post-fixed overnight. After fixation, tissue samples were processed with a sucrose gradient [10-30% (wt/vol) sucrose solution in PBS containing 0.01% (wt/vol) sodium azide and 0.02% (wt/vol) bacitracin], frozen in dry ice and preserved at -80°C until use. Tissues were sectioned into

20um thick sections with a cryostat (Microm™ HM500M/Cryostar NX70; Thermo Scientific) and collected onto SuperFrost Plus™ microscope slides (VWR International). After 2 h equilibrating at 4°C, adipose tissue sections stained with H&E (Histolab) and mounted VectaMount™ permanent mounting medium (Vector Laboratories, Inc.). Pictures were taken at 20X and 40X magnification objectives in an optical microscope (Leica). The area of SAT and VAT adipocytes (μιη²) was quantified using the ImageJ software in images obtained at 20X magnification objective.

Oil Red O ' (ORO) staining.

ORO staining was performed in formalin-fixed liver tissue from animals in protocols 2 and 3following the same above mentioned fixation and cryo sectioning procedures. After 2 h equilibrating at room temperature, slides containing liver sections were rinsed in 60% (vol/vol) isopropylic alcohol, stained in freshly prepared 0.1% (vol/vol) ORO (Sigma- Aldrich) in 60% (vol/vol) isopropylic alcohol solution for 30 min, washed in distilled water and mounted in aqueous media. Liver sections from all experimental groups and controls were immediately imaged under a 20X magnification objective using an optical microscope (Leica). Lipid visualization by ORO staining was performed in three non-consecutive liver sections separated by 100 μιη. For each section, 3 fields of view were collected in 3 individual animals per experimental group and controls.

Analysis of liver lipids.

Liver samples from animals belonging to protocol 1, 2, and 3 were frozen in liquid nitrogen and stored at -80°C until use. Hepatic triglyceride content was determined following the previously described protocol³³ for liver triglycerides determination by carcass saponification in 0.1M KOH in 99% ethanol. Triglycerides were analyzed using Free Glycerol Reagent and Glycerol Standards (Sigma- Aldrich) to construct the standard curve. The glycerol concentration (triolein equivalents) was measured by spectrophotometry

(SAFAS-MONACO™ spectrophotometer) at

and determined by extrapolation from the standard curve. Total triglyceride content was expressed in mg/g of tissue.

RNA isolation and quantitative RT-PCR.

Total RNA was isolated using the RNeasy™ Lipid Tissue Mini Kit according to the manufactures protocol (Qiagen). 500 ng of total RNA was used for cDNA preparation using the High Capacity cDNA Reverse Transcription kit (Life Technologies). Quantitative Real- Time PCR was performed in a QuantStudio™ 5 PCR system thermal cycler with Power- Up™ SYBR green PCR master mix (both Applied Biosystems). Analysis of gene expression was performed using the AACt method and relative gene expression was normalize to β-actin mRNA levels. Gene expression analyses were expressed as mRNA levels relative to Controls. Primer sequences are available upon request.

Western blotting.

For determination of circulating apoCIII, plasma was albumin depleted using

AlbuSorb™ according to the manufacture's protocol (Biotech Support Group LLC) and resuspended in RIPA buffer. Protein concentration was determined by Bradford method (BioRad). Equal amounts of protein (25μg) were loaded onto a 4-12% Bis-Tris gel

(Invitrogen). For immunoblotting, membranes were blocked with 5% (wt/vol) bovine serum albumin (BSA). After blocking, membranes were probed overnight with a rabbit anti-apoCIII antibody (Santa-Cruz). After incubation with IgG-peroxidase complexes, blots were incubated in commercial enhanced chemiluminescence reagents (ECL-Prime™; GE Healthcare), and membranes were exposed to a luminescent image analyzer (Las- 1000 Plus™; Fuji). Obtained images were quantified using ImageJ software. Ponceau Red™ (BioRad) was used to stain the membrane after reading to provide a loading control.

Statistical analysis.

For individual experiments, the number (n) is included in each figure legend within parenthesis. Data are presented as means + s.e.m. Statistical analyzes were performed with GraphPad Prism™ 5 and 7. One-way ANOVA (Tukey's post-hoc) was used. For metabolic parameters monitored using CLAMS system, the Friedman test was used as the non- parametric alternative to the one-way ANOVA with repeated measures and multiple comparisons between groups were performed by the Dunn's test. P < 0.05 was considered statistically significant. References

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Claims

We claim

1. A method for treating or limiting development of obesity, comprising administering to an obese subject, or to a subject at risk of obesity, an amount effective of an apolipoprotein CIII (apoCIII) inhibitor to reduce apoCIII expression and/or activity to control levels, thereby reducing body weight or reducing the rate of body weight increase in the obese subject or the subject at risk of obesity.

2. The method of claim 1, wherein the subject is obese.

3. The method of claim 1, wherein the subject is at risk of obesity, such as having a parent that is obese, having a sedentary lifestyle, consuming a high fat diet, having Prader- Willi syndrome or Cushing's syndrome, taking medications that lead to weight gain, including but not limited to antidepressants, anti-seizure medications, diabetes medications,

antipsychotic medications, steroids and beta blockers, being age 55 or older (55, 60, 65, 70 years of age, or older), being sleep deprived (including but not limited to having sleep apnea), and/or quitting smoking.

4. The method of any one of claims 1-3, wherein the subject is on a high fat diet, wherein the apoCIII inhibitor limits the diet-induced increase of apoCIII in the subject.

5. The method of any one of claims 1-4, wherein the subject has diabetes, such as type 2 diabetes or type 1 diabetes.

6. The method of any one of claims 1-5, wherein the treating comprises normalizing glucose tolerance and/or limiting fatty liver disease.

7. The method of any one of claims 1-6, wherein reducing body weight or reducing the rate of body weight increase comprises reducing fat levels in the subject, or reducing the increase in fat levels in the subject.

8. The method of any one of claims 1-7, wherein the apoCIII inhibitor is selected from the group consisting of anti-apoCIII antibody, anti-apoCIII aptamer, apoCIII small interfering

RNA, apoCIII small internally segmented interfering RNA, apoCIII short hairpin RNA, apoCIII microRNA, and apoCIII antisense oligonucleotides.

9. The method of any one of claims 1-8, wherein the apoCIII inhibitor comprises or consists of ASO-ISIS 353982 (AS02).

10. A method for treating fatty liver disease (FLD), NAFL, and/or NAFLD (such as

NASH), comprising administering to a subject having FLD, NAFL, and/or NAFLD (such as NASH) an amount effective of an apoCIII inhibitor to treat FLD, NAFL, and/or NAFLD (such as NASH).

11. A method for treating or limiting development of fatty liver disease (FLD), nonalcoholic fatty liver (NAFL), and/or Non-alcoholic fatty liver disease (NAFLD) (such as nonalcoholic steatohepatitis (NASH)), comprising administering to a subject at risk of FLD, NAFL, and/or NAFLD (such as NASH) an amount effective of an apoCIII inhibitor to limit development of FLD, NAFL, and/or NAFLD (such as NASH).

12. The method of claim 10 or 11 wherein the method is for treating or limiting development of NAFLD (such as NASH).

13. The method of any one of claims 10-13, wherein the apoCIII inhibitor reduces apoCIII expression and/or activity in the subject to control levels.

14. The method of any one of claims 10-13, wherein the subject has a risk factor for fatty liver disease selected from the group consisting of diabetes, obesity, insulin-resistance, a patatin-like phospholipase domain-containing 3 (PNPLA3) 148 MM variant, single- nucleotide polymorphisms (SNPs) T455C and C482T in apolipoprotein CIII (APOC3), hypertension, dyslipidemia, abetalipoproteinemia, , glycogen storage diseases, Weber- Christian disease, acute fatty liver of pregnancy, lipodystrophy, malnutrition, severe weight loss, refeeding syndrome, jejunoileal bypass, gastric bypass, jejunal diverticulosis with bacterial overgrowth, exposure to drugs or toxins (including but not limited to exposure to amiodarone, methotrexate, diltiazem, expired tetracycline, highly active antiretroviral therapy, glucocorticoids, tamoxifen- and environmental hepatotoxins (e.g., phosphorus, mushroom poisoning)), alcoholism, celiac disease, inflammatory bowel disease, HIV infection, hepatitis C infection, disparate levels of serum alanine transaminase and aspartate transaminase in the liver, and alpha 1 -antitrypsin deficiency.

15. The method of any one of claims 10-14, wherein the subject is on a high fat diet, wherein the apoCIII inhibitor limits the diet-induced increase of apoCIII in the subject.

16. The method of any one of claims 10-15, wherein the apoCIII inhibitor is selected from the group consisting of anti-apoCIII antibody, anti-apoCIII aptamer, apoCIII small interfering RNA, apoCIII small internally segmented interfering RNA, apoCIII short hairpin RNA, apoCIII microRNA, and apoCIII antisense oligonucleotides.

17. The method of any one of claims 10-16, wherein the apoCIII inhibitor comprises or consists of ASO-ISIS 353982 (AS02).

18. The method of any one of claims 10-17, wherein the method comprises one or more of decreasing liver lipid/fat content, decreasing liver inflammation, reducing the rate of increase in liver lipid/fat content, reducing the rate of increase of liver inflammation, limiting any increase/reducing incidence of cardiovascular disease and or type 2 diabetes; reducing the histologically defined NAS activity score >/= 2 ( see below) with no worsening of liver fibrosis; reducing liver fibrosis, reducing the rate of increase in liver fibrosis, reducing liver failure, and/or slowing the progression to liver failure.

19. A method for identifying a compound for treating obesity, limiting development of obesity, treating FLD, NAFL, and/or NAFLD (such as NASH), or limiting development of FLD, NAFL, and/or NAFLD (such as NASH), comprising

(a) treating a first test animal with a high fat diet;

(b) treating a second test animal with (i) the high fat diet, (ii) a test compound; and (c) determining plasma apoCIII levels and/or activity in the first test animal and the second test animal;

wherein test compounds that reduce plasma apoCIII levels and/or activity in the second test animal compared to plasma apoCIII levels and/or activity in the first test animal are candidate compounds for treating or limiting development of obesity or FLD, NAFL, and/or NAFLD (such as NASH).

20. The method of claim 19, further comprising

(d) treating a third test animal with a non-high fat diet and determining plasma apoCIII levels and/or activity in the third test animal,

wherein test compounds that lead to plasma apoCIII levels and/or activity in the second test animal similar to plasma apoCIII levels and/or activity in the third test animal are candidate compounds for treating or limiting development of obesity or FLD, NAFL, and/or NAFLD (such as NASH).

21. The method of claim 19 or 20 wherein the candidate compounds are candidate compounds for treating obesity or FLD, NAFL, and/or NAFLD (such as NASH)in a diabetic subject.

22. The method of any one of claims 19-21, wherein the test animals are mice.

23. The method of any one of claims 19-22, wherein the high fat diet comprises on a caloric basis between about 40% and about 80% fat, about 10% to about 30% carbohydrate, and about 10% to about 30% protein.

24. The method of any one of claims 19-22, wherein the high fat diet comprises on a caloric basis about 60% fat, about 20% carbohydrate, and about 20% protein.

25. The method of any one of claims 20-24, wherein the non-high fat diet comprises, on a caloric basis, about 5% to about 15% fat, about 50% to about 75% carbohydrates, and about 15% to about 40% protein.

26. The method of any one of claims 20-24, wherein the non-high fat diet comprises, on a caloric basis, about 11.4% fat, about 63% carbohydrates, and about 26% protein.