HepatosteatosisHepatosteatosisProf Samir Helmy

Assaad-KhalilUnit of Diabetes &

MetabolismAlexandria Faculty of

Medicine

2007

AgendaAgenda

Definition

Common Causes of Steatosis

Prevalence Data

Ethnic and gender differences

Relationship between fatty liver disease & Met. Syndrome

Pathogenesis of fat accumulation in the liver

Screening tests / Diagnosis

Management

DefinitionDefinition

Hepatosteatosis is defined as fat deposition in the liver that exceeds 5% of the total weight of liver, or with > 5% of hepatocytes containing fat deposits under light microscopic examination

It occurs under several disease states.

Fatty disorders of the liver may beFatty disorders of the liver may be Alcoholic (AFLD) Non-alcoholic.(NAFLD)

NAFLD includes both: Hepatic Steatosis NASH

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l

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Causes of Nonalcoholic Fatty Liver DiseaseCauses of Nonalcoholic Fatty Liver Disease

Nutritional Drugs Metabolic/Genetic Other

ObesityType 2 DMProtein-calorie

malnutritionStarvationTotal parenteral nutritionRapid weight loss

CorticosteroidsEstrogensSalicylatesCalcium channel

blockersAmiodaroneTamoxifenTetracyclineChloroquinePerhexiline maleate

Wilson's diseaseLipodystrophyDysbetalipoproteinemi

aWeber-Christian

diseaseWolman's diseaseCholesterol ester

storage

Environmental hepatotoxins

Inflammatory bowel disease

HIV* infectionSmall bowel

diverticulosis

*HIV, human immunodeficiency virus.

Inborn Errors of Metabolism Associated Inborn Errors of Metabolism Associated With SteatosisWith Steatosis

Abetalipoproteinemia Familial hepatosteatosis Galactosemia Glycogen storage disease Hereditary fructose intolerance Homocystinuria Systemic carnitine deficiency Tyrosinemia Resfum's disease Schwachman's syndrome Weber-Christian syndrome Wilson's disease

Acquired Metabolic Disorders Associated Acquired Metabolic Disorders Associated With SteatosisWith Steatosis

Diabetes mellitus Obesity Inflammatory bowel disease Jejuno-ileal bypass Kwashiorkor and marasmus Serum lipid abnormalities Starvation and cachexia Severe anemia Total parenteral nutrition

Drugs/Toxins Associated With SteatosisDrugs/Toxins Associated With Steatosis

Metals Antimony Barium salts Borates Carbon disulfide Chromates Phosphorus Rare earths of low atomic numbers Thallium compounds Uranium compounds

Cytotoxic/cytostatic drugs l-Asparaginase Azacytidine Azauridine Methotrexate

Antibiotics Azaserine Bleomycin Puromycin Tetracycline

Other drugs Amiodarone Coumadin Dichloroethylene Ethionine Ethyl bromide Estrogens Flectol H Glucocorticoids Hydrazine Hypoglycin Orotate Perhexilene maleate Safrole

Prevalence Data: Risk FactorsPrevalence Data: Risk Factors

T2 DM

28-55%Hyperlipidemia20-92%Obesity

60-95%

Distribution of NAFLD by Racial/Ethnic Distribution of NAFLD by Racial/Ethnic GroupGroup

44%

4%

29%

17%

6% 59%

10%

16%

6%

9%

NAFLD Study Population Estimated Alameda CountyPopulation (represented by KP Membership)

Caucasian African-American Latino – Hispanic Asian Other

Distribution of Gender in Persons with Distribution of Gender in Persons with NAFLDNAFLD

Number of Patients

Disease Associations in Different Racial/ Disease Associations in Different Racial/ Ethnic Groups With NAFLDEthnic Groups With NAFLD

Caucasian Latino-Hispanic

African-American

Asian P- value

BMI, mean (kg / m2)

34.0 34.0 37.8 26.7 P<0.001*

T2DM (%) 47.9 45.6 50.0 25 P=0.03* Lipids (%) 49.2 43.4 57.1 57.1 NS

* Asian versus other groups combined

* Asian versus other groups combined

Clinical features of MS in patients with Clinical features of MS in patients with NASHNASH

Yasemin H. et al., Annals of Hepatology 2006; 5(2): April-June: 109-114

Chronic hyperinsulinemia & Chronic hyperinsulinemia & carbohydrate ingestioncarbohydrate ingestion

Chronic hyperinsulinemia & carbohydrate ingestion stimulate de novo lipogenesis by stimulating the activity of lipogenic enzymes such as the sterol regulatory element binding proteins (SREBP-1c).

The FFA stored in the liver can originate from hydrolysis of dietary chylomicrons or adiposity.

SREBPs suppress IRS-2-mediated SREBPs suppress IRS-2-mediated insulin signalling in the liverinsulin signalling in the liver

Insulin receptor substrate 2 (IRS-2) is the main mediator of insulin signaling in the liver, controlling insulin sensitivity.

Sterol regulatory element binding proteins (SREBPs) directly repress transcription of IRS-2 and inhibit hepatic insulin signaling.

Ide T et al., Nat Cell Biol. 2004 Apr;6(4):351-7. Ide T et al., Nat Cell Biol. 2004 Apr;6(4):351-7.

PathogenesisPathogenesis

““2 Hit” Paradigm2 Hit” Paradigm

““Second hit” – Second hit” – Intrahepatic oxidative stressIntrahepatic oxidative stress Lipid peroxidationLipid peroxidation TNF-alpha, cytokine cascadeTNF-alpha, cytokine cascade

“ “First hit” – First hit” – Excess fat accumulationExcess fat accumulation

Oxidative Oxidative SStress & Mitochondrial Changestress & Mitochondrial Changes

Chronic oxidative stress has been implicated with a) Formation of lipid hydroperoxides.

b) Induction of certain microsomal enzymes e.g. cytochrome P450 2 El.

Mitochondrial changes: a) Mitochondrial damage with inhibition of mitochondrial

electron transport chain activity.

b) Release of mitochondrial free radicals.

c) Depletion of mitochondrial glutathione pools.

Kupffer`s Cell DysfunctionKupffer`s Cell Dysfunction

Hepatic steatosis also promotes: a) Kupffer’s cell dysfunction

b) Impaired phagocytosis

c) Chronic low grade endotoxemia.

Endotoxemia stimulate hepatic production of: a) TNF-

b) IL-6 and IL-8

c) Promote neutrophil chemotaxis & an inflammatory

response.

AdiponectinAdiponectin

Decreased serum adiponectin concentrations correlates with hepatic fat content in patients with type 2 diabetes.

Hepatocyte growth factor (HGF) is not only an antiapoptotic and antifibrotic factor of liver, but it is also an adipokine.

Serum leptin & NASH studies in humans Serum leptin & NASH studies in humans Leptin in the Field of Hepatic Fibrosis:Leptin in the Field of Hepatic Fibrosis:

A Pivotal or an Incidental PlayerA Pivotal or an Incidental Player??

CVH, chronic viral hepatitis; NASH, nonalcoholic steatohepatitis; NAFLD,nonalcoholic fatty liver disease

Sotirios K. et al Dig Dis Sci DOI 10.1007/s10620-006-9126-0

Serum Leptin Levels Are Associated With Serum Leptin Levels Are Associated With Tamoxifen-Induced Hepatic SteatosisTamoxifen-Induced Hepatic Steatosis

Serum leptin levels in patients with a normal liver or stable hepatic steatosis (group 1) and with increased hepatic steatosis (group 2)

Nazan Günel et al., Curr Med Res Opin 19(1):47-50, 2003.

Ghrelin in NAFLDGhrelin in NAFLD

Ghrelin is reduced in NAFLD vs. controls

Insulin resistance is a major factor controlling ghrelin levels in subjects with and without NAFLD.

Tumor necrosis factor-α (TNF-α) geneTumor necrosis factor-α (TNF-α) gene

Tumor necrosis factor-α (TNF-α) gene expression is increased in adipose tissue in insulin resistant obese and type 2 diabetic patients.

In patients with NASH, TNF-α gene expression is increased in both hepatocytes and adipose tissue.

The microbial theoryThe microbial theory

Conversion of choline into methylamines by microbiata in strain 129S6 on a high-fat diet reduces the bioavailability of choline and mimics the effect of choline-deficient diets, causing NAFLD.

Fatty liver due to chronic choline-deficient diet exacerbates liver hepatitis, which is predominantly facilitated by T cells.

Delayed intestinal transit may contribute to intestinal bacterial overgrowth (IBO).

Soza A et al., Dig Dis Sci. 2005 Jun;50(6):1136-40. Soza A et al., Dig Dis Sci. 2005 Jun;50(6):1136-40.

RosiglitazoneRosiglitazone

Rosiglitazone decreases liver fat and increases insulin clearance. The decrease in liver fat by rosiglitazone is associated with an increase in serum adiponectin concentrations.

Despite this beneficial effect of TZDs, in certain individuals however, in addition to increasing fat mass, TZDs have the potential to exacerbate underlying hepatosteatosis.

Hepatic ironHepatic iron

Many patients with NASH have biochemical evidence

of iron overload.

Yet, it has been suggested that the iron overload may

be a result of hemachromatosis gene mutation which

is ethnic specific and is probably not seen outside the

population of Anglo-Celtic Caucasian descent.

Uraz S et al., Dig Dis Sci. 2005 May;50(5):964-9Uraz S et al., Dig Dis Sci. 2005 May;50(5):964-9. .

Inflammatory liver steatosis caused Inflammatory liver steatosis caused by IL-12 and IL-18by IL-12 and IL-18

Acute fatty degeneration in the liver is caused by

various agents, such as aspirin, valproic acid, and

ibuprofen, that directly inhibit mitochondrial beta-

oxidation of fatty acid and oxidative

phosphorylation.

IL-12 and IL-18 may mediate inflammatory

hepatosteatosis through impairment of the

microcirculation, which leads to mitochondrial

dysfunction in hepatocytes.

Kaneda M et al., J Interferon Cytokine Res. 2003 Mar;23(3):155-62.Kaneda M et al., J Interferon Cytokine Res. 2003 Mar;23(3):155-62.

The Serum Levels of IL-1The Serum Levels of IL-1bb, IL-6, IL-8 , IL-6, IL-8 and TNF-and TNF-a a in Nonalcoholic Fatty Liverin Nonalcoholic Fatty Liver

Serum cytokine values of NAFL and control groups.

IL-8 might play a more

important role in the

pathogenesis of liver

steatosis than TNF-a,

IL-1b and IL-6.

Ülyas Tuncer et al., Turk J Med Sci 33 (2003) 381-386Ülyas Tuncer et al., Turk J Med Sci 33 (2003) 381-386

Post-transplant NASHPost-transplant NASH

Steatosis

NASH

Cirrhosis

Re-transplantation

33%

12.5%

60%

Steatosis in chronic hepatitis B : a result of Steatosis in chronic hepatitis B : a result of metabolic causes attributable to the host metabolic causes attributable to the host rather than the effect of the virusesrather than the effect of the viruses..

Comparison of cholesterol and triglyceride levels of groups with & without steatosis (P<0.05).

Comparison of BMI of groups with & without steatosis(P<0.05

Altlparmak E et al. Causes of steatosisWorld J Gastroenterol May 28, 2005 Volume 11 Number 20

NASH in patients with HCVNASH in patients with HCV

The presence of NASH in patients with HCV is strongly associated with:

Features of the metabolic syndrome Is a risk factor for advanced fibrosis with bridging.

Risk factors for advanced fibrosis in patients with NASH & HCV are:

Weight.Presence of diabetes.Presence and degree of cytological ballooning.

The liver metabolizes FFAs (partly mediated by lipase which is inhibited by insulin)Insulin

resistance (IR)

Steatosis

Lipid peroxidation

TNF

Cytokines

Iron

TGF-

Cytokines

Pathogenesis of NASHPathogenesis of NASH

FFA

Bacterial endotoxins

- Insulin signaling

- fat in liver

- TNF

When FFAs accumulate in the liver , they are oxidized by mitochondria .When FFAs accumulation exceeds oxidation ,the triglycerides and fat accumulate in the liver

Interruption of insulin signaling in hepatocytes causes fatty liver. Increase hepatic FA content causes hepatic IR

Endotoxins from intestinal bacteria escape into the mesenteric blood & trigger a sustained hepatic inflammatory cytokine response (TNF).

TNF plays an important role in IR evidenced by improvement of insulin sensitivity by genetic disruption of type 1 TNF receptors

NASH patients have higher concentration of total & FFA and total saturated & monounsaturated FAs, mainly due to the increase of: Hexadecanoic acid, Hexadecenoic acid & Octadecenoic acid. While absolute PUFA was not increased

Chronic oxidative stress with a) Induction of certain microsomal enzymes (cytochrom P450 2 El) b) Lipid peroxidation. c) Mitochondrial damage with depletion of mitochondrial

glutathion pools and release of mitochondrial free radicals which induce lipid peroxidation of hepatocyte membranes; initiate an inflammatory response with release of cytokines (TNF , IL-6, IL- 8) and stimulate fibrosis.

Steatohepatitis

Cirrhosis

Reduced antipyrine clearance (Cl-AP (reflects hepatic microsomal oxidative capacity)

Hepatic iron may contribute to the pathogenesis of NASH by : 1- Induction of lipid peroxidation of organelle membranes resulting in membrane disruption.2- Impaired mitochondrial oxidative metabolism. 3-Hepatocyte injury and death. 4- Lipocyte activation and stimulation of collagen type I gene activation & fibrosis.

Clinical Features of NASHClinical Features of NASH

Symptoms o Vague (fatigue, malaise, right upper quadrant discomfort) o Variable o Mostly absent

Signs o Hepatomegaly common o Splenomegaly in some o Portal HTN unusual

Laboratory values o Increased AST, ALT typical o +/2 increased alk. phos., GGT o Increased cholesterol, triglycerides common o Increased glucose common o Viral markers (2) o Autoantibodies (2) o Iron studies abnormal sometimes

Imaging o Fatty liver

TransaminasesTransaminases

Alanine transaminase (ALT) levels are higher than aspartate transaminase (AST) levels in most instances.

AST level may occasionally be higher than the ALT level, especially in the presence of cirrhosis.

The usual AST/ALT ratio is < 1 in patients with NASH and may be used to differentiate it from alcoholic liver disease (> 2 in the latter).

NASH can present with normal ALT values (in 20-25% of patients) despite the presence of a full histologic spectrum.

Symptoms of obstructive sleep apnea Symptoms of obstructive sleep apnea in patients with nonalcoholic fatty liverin patients with nonalcoholic fatty liverdiseasedisease

Approximately one-half of NAFLD patients, whether NAFL or NASH, have Syndrome of Sleep Apnea.

Singh H et al., Dig Dis Sci. 2005 Dec;50(12):2338-43.Singh H et al., Dig Dis Sci. 2005 Dec;50(12):2338-43.

Demonstration of fat in Demonstration of fat in the liver, staging & gradingthe liver, staging & grading

The presence of fat in the liver can be demonstrated by various imaging modalities; however, no current noninvasive method can distinguish NASH from NAFLD.

Liver biopsy remains the gold standard for staging and grading

Histologic criteriaHistologic criteria

There are 2 histologic patterns of NASH:

Steatosis.

Steatohepatitis

The principle histologic features of NASH are:

The presence of macrovesicular fatty change in hepatocytes

Displacement of the nucleus to the edge of the cell.

Leucocytic infiltration in steatohepatitis.

ImagingImaging

Transabdominal ultrasound is a sensitive, noninvasive method for detecting NAFLD. However, diagnostic criteria are highly operator-dependant and non-standardized

Spleen-minus-liver attenuation difference (DeltaS-LA) derived from CT shows a good correlation with the pathology of hepatosteatosis.

Duman DG et al.,Duman DG et al., Dig Dis Sci. 2006 Feb;51(2):346-51. Dig Dis Sci. 2006 Feb;51(2):346-51.

ScintigraphyScintigraphy

Using scintigraphy, a strong, significant inverse correlation between the severity of steatosis, hepatic triglycerides content, and 99mTc-mebrofenin uptake rate was observed.

In the future, noninvasive "dynamic" breath tests may disclose specific alterations in metabolic pathways.

Proposed Histologic Spectrum NAFLDProposed Histologic Spectrum NAFLD

Fat+

Inflammation

FatInflammation

BallooningDegeneration

FatBallooning

DegenerationFibrosis

+/-Mallory Bodies

FAT

Stage I

Stage IIStage III

Stage IV

Matteoni et al, Gastroenterol 1999Examples of non-alcoholic fatty liver disease (NAFLD)

NASH as a Cause of End-Stage Liver NASH as a Cause of End-Stage Liver DiseaseDisease

Primary indication for OLT in 31/1,207 (2.6%) of patients evaluated at Mayo between 1993-98.

16/546 (2.9%) underwent transplantation for end-stage NASH

Charlton et al. Liver Transpl, 2001

OLT= Organ Liver Transplant

Current Management & Current Management & Future Therapy for NASHFuture Therapy for NASH

Current Management Potential Future Therapy

Weight loss Treatment of diabetes &

lipid disorders Avoid Ethanol & hepato-

toxic drugs

Constraining macrophage activation Antioxidants (vitamin E, glutathione

pro-drugs) Antibiotics (gut decontamination) Anti-cytokines (anti-TNF

antibodies, soluble receptors) Protecting hepatocyte ATP stores

PPAR agonists Minimizing Cyp2E1 activity

Dietary modification (avoid fats)

NASH and effect of weight lossNASH and effect of weight loss

Histological & laboratory improvement occurs with a 10% decrease in body weight.

However, in some patients, rapid weight loss may result in mild increase in inflammatory lesions (hepatitis), despite the regression of steatosis.

This may result from rapid mobilization of fatty acids or cytokines from adipose tissue, especially visceral fat.

SilymarinSilymarin

Therapeutic approaches include silymarin, the seed extract of milk thistle, which is a mixture of flavonolignans

A hepatoprotective agent stimulating secretion of adiponectin.

This flavanoid has also antioxidant, antifibrotic, and membrane-stabilizing effects.

Schuppan D et al., Hepatology 1999;30:1099-1104

Vitamin E & BetaineVitamin E & Betaine

Vitamin E, an antioxidant agent, reduces liver inflammation and necrosis.

Betaine, a naturally occurring choline metabolite, improves both biochemical parameters and liver histology.

MetforminMetformin

Metformin improves biochemical indices of hepatocellular injury and insulin resistance

Long-term metformin (500 mg 3 times /day for 4 months) resulted in:

Reduced transaminase levels, which returned to normal in 50% of actively-treated patients.

Improved insulin sensitivity

Decreased liver volume by 20%.

Mechanism of action of metformin in NASH: Inhibits the hepatic TNF- activity . Inhibits the expression of UCP-2 .

PPAR AgonistsPPAR Agonists

Thiazolidinediones reduce hepatic fat stores in type 2 diabetes mellitus.

PPAR-alpha agonists exert antioxidative effects by inducing antioxidant enzymes, in addition to diminishing steatosis

Insulin therapyInsulin therapy

Insulin therapy improves hepatic insulin sensitivity

It slightly but significantly reduces liver fat content, independent of serum adiponectin.

Patients receiving long-term total Patients receiving long-term total parenteral nutritionparenteral nutrition

Patients receiving long-term total parenteral nutrition may develop NAFL partially because of choline deficiency.

Besides, bacterial overgrowth is enhanced in the resting intestines.

Choline supplementation has been reported to improve or revert hepatic steatosis.

Buchman AL et al., Gastroenterology 1992; 102:1363-1370 Buchman AL,et al. Hepatology 1995;22:1390-1403 Pappo I et al., J Clin Res 1991;51:106-112 Freud HR rt al., J Surg Res 1985;38: 356-363

Iron depletionIron depletion

Hepatic iron overload is frequent in NAFLD, and iron depletion improves liver function tests and insulin resistance.

The addition of iron depletion therapy to standard diet/exercise reduces serum ALT levels in NASH independently from iron stores, thus representing a promising effective, safe, and low-cost therapy.

Glucagon-like peptide-1Glucagon-like peptide-1

Glucagon-like peptide-1 (GLP-1), reverses hepatic steatosis.

Fat-reduced dietFat-reduced diet

Protecting hepatocytes ATP stores & inhibiting

Cyp2E1 activity by dietary modifications (fat-

reduced diet), may be beneficial in NAFLD patients.

Chavin K et al., J Biol Chem 1999;274:5692-5700 Cortez-Pinto H et al., JAMA 1999;282:1659-1664 Weltman MDet al. Hepatology 1998;27:128-133

Surgical treatment (gastroplasty) improves Surgical treatment (gastroplasty) improves metabolic abnormalities & hepatic lesions of metabolic abnormalities & hepatic lesions of NAFLD in long-term observationsNAFLD in long-term observations

Diffuse grade 3 steatosis & steatohepatitis in liver biopsy taken during gastroplasty. H&E ×250.

Significant improvement of histologic lesions 8 months after gastroplasty. H&E ×250.

K. JASKIEWICZ et al. , Digestive Diseases and Sciences, Vol. 51, No. 1 (2006), pp. 21–26

Take Home MessageTake Home Message

The main risk factors for steatosis are obesity, type 2 diabetes & dyslipidemia

Ethnic & gender differences have important implications for the development of steatosis-related liver disease.

Met. syndrome & adiponectin concentrations are independently associated with the probability of steatosis

Intestinal bacterial overgrowth has been suggested to play a pathogenic role in patients with nonalcoholic fatty liver disease (NAFLD).

Hepatic iron may contribute to the pathogenesis of NASH

IL-12 and IL-18 may mediate inflammatory hepatosteatosis

Approximately one-half of NAFLD patients, have SOSA.

The most common abnormality in liver function tests is a two- to fivefold elevation in ALT and AST

Liver biopsy remains the gold standard for staging and grading

Transabdominal ultrasound is a sensitive, noninvasive method for detecting NAFLD. However, diagnostic criteria are highly operator-dependant and non standardized

Take Home MessageTake Home Message

Current management includes weight loss, treatment of diabetes and lipid disorders as well as avoidance of alcohol and hepatotoxic drugs.

Weight loss (about 10%) improves biochemical & histological profiles in the majority of obese NASH patients

Potential future therapies include constraining macrophage activation using antioxidants, the use of antibiotics, anti-cytokines & PPAR agonists. Lastly minimizing Cyp2E1 activity by dietary modification.

Iron depletion therapy reduces serum ALT levels in NASH & may be a promising low cost, future therapy.

Take Home MessageTake Home Message

Thank Thank YouYou ! !