Along with its anti-obesity effects, a new study has shown that …

Mulberry Can Protect
Your Liver

Fatty liver disease that develops in the absence of alcohol abuse
(non-alcoholic fatty liver disease) is recognized increasingly as
a major health burden
By Will Block

Y

ou can’t be healthy if your liver does not live up to the purposes for which it evolved. That’s because your liver is a vital organ with a wide range of functions, including detoxification, production of the biochemicals necessary for digestion, and protein synthesis. Simply put, the liver is necessary for survival. Besides, there is currently no way to compensate for the absence of liver function in the long term—despite the possibility of dialysis, which can only be done for a short while—so transplants are the bottom line of a failing liver. Bear in mind … a transplantable liver is hard (and expensive) to obtain. Oh yes, survivability has increased for those who are fortunate enough to obtain one.

Jobs’ Liver Transplant

Take Steve Jobs. Two years ago, he got a liver transplant to prolong his life. Apparently his pancreatic cancer had damaged his liver. To get the liver, Jobs journeyed to Tennessee, which unlike Northern California didn’t have a long waiting list and where there were more livers to go around. Unfortunately, many of the Californians who needed livers couldn’t get them for a number of reasons. First, they didn’t have the wherewithal or know-how of a Jobs. Secondly, they didn’t have the dough to travel around the country, undergo the extensive evaluations at multiple transplant centers, and insure their readiness within hours of when the next liver became available. But good for Jobs! He has certainly earned our gratitude and added a lot of beauty and productivity to our lives.

Currently, about 17,000 adults and children who have been medically approved for liver transplants are waiting for donated livers to become available.1 The waiting list grows every year. But a liver transplant costs up to $500,000, and anti-rejection medicines need to be taken each day throughout the patient’s life. Those expenses add $2,000 to $5,000 a month to the cost of being the recipient of a transplant.

Back to “reality” of not needing a transplant, the liver plays a major role in metabolism, including glycogen storage, decomposition of red blood cells, plasma protein synthesis, hormone production, and detoxification (as mentioned). Located below the diaphragm in the abdominal-pelvic region of the abdomen, this dynamic organ produces bile, an alkaline compound which aids in digestion via the emulsification of lipids. The liver’s highly specialized tissues regulate a wide variety of high-volume biochemical reactions, including the synthesis and breakdown of small and complex molecules, many of which are necessary for vital functions.

The Commonality of Non-Alcoholic Fatty Liver Disease

Fatty liver disease, which develops in the absence of alcohol abuse (non-alcoholic fatty liver disease or NAFLD), is increasingly recognized as a major health burden. In fact, approximately 20–30% of adults are estimated to have excess liver fat accumulation in a normal population.2 Many researchers think that liver disease is inevitably connected to the excessive consumption of alcohol. This isn’t true. In fact, NAFLD is one of the most common metabolic syndromes and is strongly associated with obesity and insulin resistance.3 Diabetic beware!


In fact, approximately 20–30% of
adults are estimated to
have excess liver fat accumulation in
a normal population.


NAFLD is characterized by high hepatic triglycerides (TG) resulting from an imbalance between uptake, synthesis, export, and oxidation of fatty acids.4 Indeed, increased free fatty acids (FFA) supplied to the liver play a major role in the early stage of NAFLD. Studies have shown that sterol regulatory element-binding proteins (SREBPs) regulate lipid metabolism.5 The transcription factor SREBP-1c is essential to making fatty acids and synthesizing TG, while SREBP-2 regulates genes involved in cholesterol biosynthesis, especially low-density lipoprotein receptor, 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, and SREBP-2 itself.6,7

In addition, it has been shown that peroxisome proliferator-activated receptors (PPARs) (see “Cinnamon Swoops Down to Retard Diabetes” in the May, 2010 issue) intervene in the regulation of genes associated with balancing lipid production (homeostasis).8 In mice in which PPARa* activity has been nullified, a chronically high-fat diet has produced the development of severely fatty livers, owing to their diminished hepatic β-oxidation captivity. Medical terms that related to the liver are often prefixed with hepato- or hepatic from the Greek word for liver (he–par hpar).


*PPARα is a specific member of a group of nuclear receptor proteins that function as transcription factors regulating the expression of genes, in liver, kidney, heart, muscle, fat tissue, and elsewhere.



The data suggest that MWE lessens
hepatic lipid accumulation through
the expression of TG biosynthesis and
fatty acid β-oxidation.


Recent evidence has shown that adenosine monophosphate (AMP)-activated protein kinase (AMPK) plays a key role in energy homeostasis and it has been proposed to monitor changes in the energy status of cells.10

The Mulberry Inhibits TG in the Liver Through AMPK

Mulberries (Morus alba L.) are widely distributed in Asia where they have been used as a traditional food and found to possess pharmacological benefits such as liver protection, eyesight improvement, blood pressure reduction, and helping to prevent cardiovascular disease. Dietary mulberry (as fruit) has been reported to have antihyperlipidemic,11 antidiabetic,12 and antioxidative effects.13,14 These properties are due to mulberry’s constituents, including high levels of anthocyanins and polyphenolics, which have many biochemical activities, including antioxidant activities and antitumor properties (see “A Newly Rediscovered Anti-Obesity Supplement” in the September, 2011 issue).


Because the accumulation of
cholesterol is observed in
late-stage NAFLD, it is possible that
MWE has other effects in
improving atherosclerosis.


Previous studies suggest that mulberry water extract (MWE) play a role in protecting against hyperlipidemia in hamsters when supplementing MWE in their high-fat/cholesterol diet.15 In a new study, researchers from the Institute of Biochemistry and Biotechnology, College of Medicine, Chung Shan Medical University, Taichung, Taiwan and other divisions of Chung Shan investigated how MWE can inhibit hepatic TG accumulation through activation of AMPK and how MWE affects lipid metabolism in human hepatoma (cancer of the liver) HepG2 cells.16 This extends what has been previously known about how MWE works.

MWE was prepared from the fruit of M. alba L., which was macerated, stirred with water filtered, centrifuged, and concentrated under reduced pressure. The aqueous extract was then lyophilized (freeze-dried) to obtain the MWE. Then a cell line proven to be a useful model of the human liver cell—HepG2—because of its high proportion of liver-specific proteins—was exposed to oleic acid (OA), a monounsaturated fatty acid known to induce cell apoptosis and reduce cell viability. To this was added MWE to determine its effects on cell viability of the OA exposed human HepG2 cells. MWE treatment of HepG2 cells resulted in slight inhibition of those cells indicating that the various concentrations of MWE tested were not cytotoxic to HepG2 cells.

MWE Protects Against Fatty Liver

LEM1112graph260.gif
Fig. 1
(click on thumbnail for full sized image)
Furthermore, upon analyzing the development of lipid accumulation in an in vitro model of hepatic steatosis—the abnormal retention of lipids within a cell, reflecting impairment of the normal processes of synthesis and elimination of triglyceride fat—lipid accumulation was evident in all cells exposed to OA. The intracellular lipid content was reduced significantly by treatment with MWE. While OA treatment alone caused a significant increase in lipid accumulation by 100%, these levels were reduced to 69.7% and 63% in 2–3 mg/mL concentration of MWE, respectively, as compared to OA treatment and control (C). (See Fig. 1.)

MWE Stimulates AMPK Which Decreases ACC, and Suppresses A-FABP Expression in HepG2 Cells

The cited studies indicate that AMPK has been implicated as a key regulator of energy homeostasis, including glucose transport, gluconeogenesis, and lipolysis. In investigating the effects of MWE on the phosphorylation of AMPK, HepG2 cells were treated with OA for 24 hours. MWE stimulated phosphorylated AMPK at concentrations of 2–3 mg/mL. The enzyme acetylcoenzyme A carboxylase (ACC), which is involved in fatty acid synthesis, has been identified as the primary target of AMPK.

The researchers also investigated the decrease of ACC in MWE treatment of HepG2 cells. It has been reported that fatty acid synthase (FAS) is a key enzyme in lipogenesis, and that FAS and other enzymes are transcriptionally regulated by SREBP-1c. In their analysis, it was shown that FAS and SREBP-1 protein levels were reduced significantly under MWE treatment. Also, the effects of MWE on the expression of adipocyte fatty acid binding protein (A-FABP) suppressed A-FABP expression in HepG2 cells.

MWE Suppresses HMG-CoA and SREBP-2 in HepG2 Cells

There followed further evaluation of whether MWE lessens lipid accumulation through regulation of cholesterol synthesis. HMG-CoA reductase is the rate-limiting enzyme in cholesterol biosynthesis, and SREBP-2 targets the enzymes of cholesterol biosynthesis. The findings reveal that suppression of HMG-CoA reductase and SREBP-2 in HepG2 cells was detected after treatment with 2–3 mg/mL MWE, respectively, as compared to OA treatment.

MWE Reduces GAPT, an Enzyme Involved in TG Synthesis

Moreover, MWE treatments increase AMPK phosphorylation. When cells were co-treated with OA and various concentrations of MWE for 24 hours, AMPK phosphorylation was detected. Glycerol-3-phosphate acyltransferase (GPAT), an enzyme involved in TG and phospholipid synthesis, catalyzing the first committed step of de novo TG synthesis. In TG biosynthesis, the protein expression of GAPT was reduced by addition of MWE. Besides, the largest reduction of GAPT was detected in the presence of 3 mg/mL MWE.


Administration of powdered mulberry
fruit to rats on a high-fat diet resulted
in a significant decline in …

  • levels of liver triglycerides
  • total cholesterol
  • low-density lipoprotein cholesterol
  • the atherogenic index

while significantly increasing …

  • high-density lipoprotein cholesterol.

MWE through PPARα and CPT-I, Induces Fatty Acid β-Oxidation

Similarly, the expression of PPARα and carnitine palmitoyltransferase 1 (CPT-I), responsible for inducing fatty acid β-oxidation, were increased by MWE treatment in a dose-dependent manner. This suggests that MWE lessens hepatic lipid accumulation through the expression of TG biosynthesis and fatty acid β-oxidation.

MWE Possesses a Hepatic Hypolipidemic Mechanism

To sum this all up (if you really want to understand this, please read the past few sections again), the hepatic hypolipidemic mechanism of MWE is highly related to expression of lipogenic enzymes (SREPB-1, FAS, ACC, and A-FABP), cholesterol biosynthesis (SREBP- 2 and HMG-CoA reductase), TG biosynthesis (GPAT), and fatty acid β-oxidation (PPARα and CPT-1) in HepG2 cells.

The strength of these conclusions is enhanced because HepG2 cells are significantly similar to an intact liver: they possess the lipoprotein-mediated uptake and secretion of cholesterol, and its metabolism to bile acids. Indeed, the lipid composition of HepG2 cells is close to that of normal human liver cells. The HepG2 cell line has been studied under a variety of different conditions with Wang et al suggesting that the HepG2 cell line offered an alternative and reliable model for studies on liver lipid metabolism.17 This adds to the researchers reasons for choosing the HepG2 cell line to assay the impact of MWE on lipid metabolism.

MWE Inhibits Liver Lipid Accumulation

The important finding of the study shows that MWE inhibited cellular lipid accumulation through the activation of AMPK and suppression of the lipogenic enzyme. In fact, at least one other study has shown that phytochemicals can activate AMPK and that this can lead to regulation of a number of downstream targets involved in lipid metabolism in order to maintain energy homeostasis.18

Among the AMPK downstream targets, HMG-CoA reductase and ACC have been well identified. ACC is an important enzyme for synthesis of malonyl-CoA, which inhibits CPT-1, a transporter of long-chain fatty acyl groups into the mitochondria to undergo &beta-oxidation.

Other reports have also suggested that suppression of ACC by AMPK phosphorylation leads to the fall in malonyl-CoA content, the subsequent decrease in fatty acid synthesis and increase in fatty acid oxidation through the regulation of CPT-1. In the present study, MWE treatments increased CPT-1 expression and AMPK phosphorylation, and decreased ACC protein expression. This suggests that activation of AMPK by MWE inhibits the protein expression of ACC, increases CPT-1 levels, and results in the stimulation of fatty acid oxidation.

The point remains that NAFLD is characterized by excessive accumulation of lipids in hepatocytes, mostly TG and FFA, causing cell injury and death. A diagnosis of NAFLD should be strongly suspected in patients in the presence of atherosclerosis and liver-related disease. Because the accumulation of cholesterol is observed in late-stage NAFLD, it is possible that MWE has other effects in improving atherosclerosis.


The research findings might be
beneficial to various treatment
strategies for fatty livers and obesity.


Lipid accumulation in liver may be caused by enhanced de novo lipogenesis, activation of lipid uptake, and lowering of lipid catabolism. FAS and GPAT are key enzymes in de novo fatty acid and TG synthesis in mammals. SREBP-1 is well known as the transcription factor regulating the gene expression of these lipogenic enzymes in the liver.

In the current study, expressions of FAS, GPAT, and SREBP-1 were changed by co-treatment with OA and MWE. These results indicate that lipid accumulation in OA and MWE-treated HepG2 cells is associated with decreased expression of SREBP-1 and its downstream lipogenic genes. Numerous studies have indicated that activation of AMPK effectively suppresses the expression of SREBP-1 in the liver. Consequently, these findings suggest that the ability of MWE to suppress FAS expression may occur through AMPK activation and suppression of SREBP-1 in HepG2 cells.

It is also known that PPARs mediate lipid homeostasis. In fact, PPARα is expressed at high levels in the liver, kidney, and heart where it leads to peroxisomal β-oxidation. Decreased mitochondrial fatty acid oxidation has long been considered to be the major mechanism underlying disturbances in lipid accumulation in liver, and in the current study, MWE significantly increased the expression of PPARα and CPT-1, affecting fatty acid β-oxidation in HepG2 cells. Other studies have indicated that polyphenols, lipids, and polysaccharides have numerous biological properties, including hypolipidemic effects. Thus the researchers assumed that MWE—containing polyphenols, lipids, and polysaccharide—might enable lipid-lowering effect, and they appear to have been right.

Anticipating Study

The results of the Taiwanese study were not completely unanticipated. A Chinese study of a year earlier investigated the phytochemical constituents of a freeze-dried powder of mulberry (Morus alba L.) fruit (MFP) for its hypolipidemic and antioxidant effects as a dietary supplement in rats who were fed 4 weeks of either a high-fat or a normal diet supplemented with 5% or 10% MFP.19 Administration of MFP to rats on a high-fat diet resulted in a significant decline in levels of serum and liver triglyceride, total cholesterol, serum low-density lipoprotein cholesterol, and a decrease in the atherogenic index, while the serum high-density lipoprotein cholesterol was significantly increased.


Thus, in the worst case, it is possible
that MWE might put the brakes on
the downward spiral for
the need of a liver transplant.


In addition, they also found that the serum and liver content of a lipid peroxidation product, significantly decreased, while the superoxide dismutase (SOD) of red blood cell and liver, as well blood glutathione peroxidase (GSH-Px) activities significantly increased. No significant changes in lipid profile in the serum and liver were observed in rats on a normal diet supplemented with MFP, but blood and liver antioxidant status improved, as measured by SOD and GSH-Px activity, and lipid peroxidation was reduced. These beneficial effects of MFP on hypolipidemic rats might be attributed to its dietary fiber, fatty acids, phenolics, flavonoids, anthocyanins, vitamins and trace elements content the Chinese researchers thought.

Mulberry Water Extract for Maintaining Proper Liver Function

In summary, the Taiwanese researchers provide evidence that MWE is likely to play a significant role in reducing HepG2 cellular lipid accumulation by increasing AMPK phosphorylation, thereby inhibiting the expression of SREBP-1, FAS, and ACC, and further suppressing TG and cholesterol synthesis. The results contribute new understanding to how natural products such as polyphenol-rich MWE can affect lipid metabolism in vitro. In seems likely that MWE, because of its low toxicity, is a grand candidate for in vivo studies. These findings might be beneficial to various treatment strategies for fatty livers and obesity in the future. Also, remember that the researchers chose human hepatoma (cancer of the liver) HepG2 cells for their study. Thus, in the worst case, it is possible that MWE might put the brakes on the downward spiral for the need of a liver transplant. In the less-than-best case, MWE can help keep your liver healthy, working in the service of your life.

References

  1. More about organ donation. American Liver Foundation. Updated: http://www.liverfoundation.org/patients/organdonor/ about/October 4th, 2011 Accessed October 23, 201.
  2. Neuschwander-Tetri BA, Caldwell SH. Nonalcoholic steatohepatitis: summary of an AASLD Single Topic Conference. Hepatology 2003;37:1202–19.
  3. Postic C, Girard J. Contribution of de novo fatty acid synthesis to hepatic steatosis and insulin resistance: lessons from genetically engineered mice. J Clin Invest 2008;118:829–38.
  4. Tamura S, Shimomura I. Contribution of adipose tissue and de novo lipogenesis to nonalcoholic fatty liver disease. J Clin Invest 2005;115:1139–42.
  5. Brown MS, Goldstein JL. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell 1997;89:331–40.
  6. Horton JD, Shimomura I, Brown MS, Hammer RE, Goldstein JL, Shimano H, Activation of cholesterol synthesis in preference to fatty acid synthesis in liver and adipose tissue of transgenic mice overproducing sterol regulatory element-binding protein-2. J Clin Invest 1998;101:2331–9.
  7. Hua X, Yokoyama C, Wu J, Briggs MR, Brown MS, Goldstein JL, et al. SREBP-2, a second basic-helix-loop-helix-leucine zipperprotein that stimulates transcription by binding to a sterol regulatory element. Proc Natl Acad Sci 1993;USA 90:11603–7 .
  8. Lee CH, Olson P and Evans RM. Minireview: lipid metabolism, metabolic diseases, and peroxisome proliferator-activated receptors. Endocrinology 2003;144:2201–7.
  9. Kersten S, Seydoux J, Peters JM, Gonzalez FJ, Desvergne B, Wahli W. Peroxisome proliferator-activated receptor alpha mediates the adaptive response to fasting. J Clin Invest 2999;103:1489–98.
  10. Carling D. AMP-activated protein kinase: balancing the scales. Biochimie 2005;87:87–91.
  11. El-Beshbishy HA, Singab AN, Sinkkonen J and Pihlaja K. Hypolipidemic and antioxidant effects of Morus alba L. (Egyptian mulberry) root bark fractions supplementation in cholesterol-fed rats. Life Sci 200678:2724–33.
  12. Kimura T, Nakagawa K, Kubota H, Kojima Y, Goto Y, Yamagishi K, et al. Food-grade mulberry powder enriched with 1-deoxynojirimycin suppresses the elevation of postprandial blood glucose in humans. J Agric Food Chem 2007;55:5869–74.
  13. Isabelle M, Lee BL, Ong, CN, Liu X, Huang D. Peroxyl radical scavenging capacity, polyphenolics, and lipophilic antioxidantprofiles of mulberry fruits cultivated in southern China. J Agric Food Chem 2008;56:9410–6.
  14. Andallu B, Suryakantham V, Lakshmi Srikanthi B, Reddy GK. Effect of mulberry (Morus indica L.) therapy on plasma and erythrocyte membrane lipids in patients with type 2 diabetes. Clin Chim Acta 2001;314:47–53.
  15. Liu LK, Chou FP, Chen YC, Chyau CC, Ho HH, Wang CJ. Effects of mulberry (Morus alba L.) extracts on lipid homeostasis in vitro and in vivo. J Agric Food Chem 2009;57:7605–11.
  16. Ou TT, Hsu MJ, Chan KC, Huang CN, Ho HH, Wang CJ. Mulberry extract inhibits oleic acid-induced lipid accumulation via reduction of lipogenesis and promotion of hepatic lipid clearance. J Sci Food Agric 2011 Oct 17. doi: 10.1002/jsfa.4489. [Epub ahead of print]
  17. Wang SR, PessahM, Infante J, Catala D, Salvat C, Infante R. Lipid and lipoprotein metabolism in HepG2 cells. Biochim Biophys Acta 1988;961:351–63.
  18. Hwang JT, Park IJ, Shin JI, Lee YK, Lee SK, Baik HW, et al. Genistein, EGCG, and capsaicin inhibit adipocyte differentiation process via activating AMP-activated protein kinase. Biochem Biophys Res Commun 2005;338:694–9.
  19. Yang X, Yang L, Zheng H. Hypolipidemic and antioxidant effects of mulberry (Morus alba L.) fruit in hyperlipidaemia rats. Food Chem Toxicol 2010 Aug-Sep;48(8-9):2374-9.


Will Block is the publisher and editorial director of Life Enhancement magazine.

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