Hepatic Copper Regulates Lipid Metabolism in High Fat Diet Fed Mice

Alexandra Lund Author

07/27/2021 Added

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A study on lipid metabolism in hepatic Copper Transporter 1 knockout mice

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[00:00:00.300]Hello. My name is Alexandra Lund,
[00:00:02.220]and I am a University of Nebraska SRP student in the Redox Biology REU.
[00:00:07.290]This summer, I worked in Dr. Lee's
[00:00:08.990]lab in the UNL Redox Biology Center studying how hepatic copper
[00:00:13.290]regulates lipid metabolism in high-fat diet fed mice.
[00:00:17.400]As an introduction,
[00:00:18.540]copper is an important trace metal for living organisms because of its ability
[00:00:22.260]to participate in redox reactions.
[00:00:24.570]Basically, this means that a wide variety of enzymes need copper to function properly and process energy, nutrients,
[00:00:31.320]and information. Because of this, copper homeostasis or balance is incredibly
[00:00:36.120]important for organismal health.
[00:00:38.310]The main hub for copper processing and balance is the liver.
[00:00:41.670]Copper enters hepatocytes or liver cells via Copper Transporter 1 or
[00:00:46.140]CTR1. Once in the cell,
[00:00:48.690]copper is processed in a variety of ways.
[00:00:51.250]In the liver, which also processes a majority of the body's nutrients in addition to copper,
[00:00:56.340]copper imbalance has been linked specifically to obesity and non-alcoholic fatty liver disease
[00:01:02.124]or NAFLD. Obesity and NAFLD are both
[00:01:05.940]significant health problems whose causing factors are still relatively unknown.
[00:01:10.410]In response to this need,
[00:01:12.080]Dr. Lee's lab created a mouse with CTR1 removed from hepatocytes to test
[00:01:17.190]cellular functions under copper deficient conditions.
[00:01:20.070]They discovered that CTR1 knockout in hepatocytes generally leads to an
[00:01:24.180]overall decrease in high-fat diet induced weight gain. Currently,
[00:01:28.560]we believe this occurs because when CTR1 is decreased,
[00:01:31.950]copper becomes deficient in cells.
[00:01:35.430]This deficiency induces oxidative,
[00:01:37.770]mitochondrial and other unknown stressors.
[00:01:41.100]The stress, hypothetically, is enough to induce improper lipid metabolism or
[00:01:45.900]lipid dysregulation.
[00:01:47.550]This dysregulation can result in lipid toxicity and eventually hepatic or
[00:01:52.350]metabolic diseases, including obesity and NAFLD.
[00:01:56.490]This leads me directly to my research question for the summer:
[00:01:59.790]How does hepatic copper affect lipid metabolism?
[00:02:02.770]In other words, I was asking the question: How do copper levels in the liver change the way
[00:02:07.920]organisms process fats?
[00:02:10.090]To answer this question, this summer,
[00:02:11.670]I measured lipid and liver health markers in the liver and serum of CTR1
[00:02:16.260]knockout, and control or wild type mice. As mentioned before,
[00:02:20.030]Dr. Lee's lab has previously created a CTR1 knockout mouse model to study the
[00:02:24.870]connection between copper homeostasis and metabolic disorders.
[00:02:28.500]The CTR1 flox flox mouse is the wild type mouse,
[00:02:31.740]meaning, it has the characteristics or phenotype of a normal mouse.
[00:02:35.850]The CTR1 flox flox albumin Cre mouse is the knockout mouse.
[00:02:40.080]This mouse was created to have a liver specific CTR1 knockout,
[00:02:43.860]meaning, the livers of these mice do not contain CTR1, that important
[00:02:48.120]copper transporter protein.
[00:02:49.800]This system basically induces copper deficiency in the livers of affected mice.
[00:02:54.780]These mice were analyzed under three different conditions.
[00:02:58.020]The first condition was a chow diet condition,
[00:03:01.360]which basically means that the mice ate normal food.
[00:03:04.240]The second condition was a one week high-fat diet condition.
[00:03:07.660]This basically means that the mice ate a high fat diet for one week.
[00:03:11.290]The third condition was a four weeks high-fat diet,
[00:03:13.810]which means the mice ate a high-fat diet for four weeks.
[00:03:18.190]The mouse tissue was then collected and analyzed for different lipid or
[00:03:23.050]fat levels, liver health markers, and cholesterol.
[00:03:26.560]The data was then consolidated into bar graphs
[00:03:28.900]comparing levels between wild type and Cre mice,
[00:03:32.590]and also between the three different groups.
[00:03:35.470]These first graphs show the overall health of the livers collected from the mice.
[00:03:39.580]Throughout these graphs,
[00:03:40.840]an asterisk indicates a significant change between the wild type or control
[00:03:45.280]mouse and the Cre or knockout mouse.
[00:03:47.710]A pound symbol indicates a change value from the control treatment or chow diet.
[00:03:53.320]And an addition sign indicates a significant change between the one and four
[00:03:57.010]weeks treatments.
[00:03:58.720]As mentioned before, these graphs show overall liver health measured by ALT and
[00:04:03.310]AST, which are markers used to determine overall level health.
[00:04:07.570]Generally, the higher these markers are the more liver damage has occurred.
[00:04:12.100]As you can see,
[00:04:13.150]both AST and ALT are significantly increased in the Cre or knockout mice
[00:04:18.130]after both one and four weeks high-fat diet.
[00:04:21.580]This indicates that there is a significant connection between copper deficiency,
[00:04:25.720]overall liver health, and lipid metabolism.
[00:04:28.720]However, this led us to wonder what specifically is happening in these knockout
[00:04:32.890]mice eating a high-fat diet that would cause this type of damage.
[00:04:37.030]First, we tested two different lipids or fats important to the body: triglycerides and
[00:04:41.710]free fatty acids.
[00:04:43.240]We found that free fatty acids displayed no particularly significant changes
[00:04:47.680]over the course of the treatments. However,
[00:04:49.930]triglyceride levels did show a significant decrease after four weeks high-fat
[00:04:54.400]diet, reinforcing our hypothesis that copper alters lipid metabolism.
[00:04:59.710]Next, we tested cholesterol levels in our mice and found some very interesting results.
[00:05:04.808]As you can see, the wild type mice display,
[00:05:06.940]a marked cholesterol increase between the chow diet,
[00:05:10.240]the one week high-fat diet and the four weeks high-fat diet.
[00:05:13.420]This increase is the normal physiological response to increased lipid intake.
[00:05:18.340]However, the Cre, or knockout mice, did not display the same increase.
[00:05:22.930]This leads to the drastic decrease between the Cre mice and their wild
[00:05:27.790]type counterparts in both the one week and four week treatment groups.
[00:05:31.870]Additionally, serum cholesterol levels show a similar pattern.
[00:05:35.680]Although overall more stable,
[00:05:37.480]the knockout cholesterol show a marked decrease when compared to their wild type
[00:05:41.680]counterparts for the four week treatment group.
[00:05:44.740]The data from our further serum analysis of lipoproteins, or cholesterol carrying
[00:05:49.030]elements in the blood, matched with this conclusion. As you can see,
[00:05:53.140]Cre mice have significantly decreased lipoprotein levels after four weeks high-fat diet.
[00:05:58.820]Overall, this data suggests that in CTR1 knockout mice,
[00:06:02.270]a high-fat diet induces two major responses. The first is liver damage,
[00:06:07.010]which is shown by the significantly increased ALT and AST markers.
[00:06:11.270]The second response is decreased cholesterol levels in both hepatic tissue and
[00:06:15.980]serum of knockout mice.
[00:06:18.230]Although we do not know the specific reason these results occur yet,
[00:06:21.290]we do have a few hypotheses.
[00:06:23.240]It is possible that increased lipid intake triggers
[00:06:25.880]lipotoxicity, or cell damage caused by lipids, in knockout hepatocytes
[00:06:30.230]because of the combined oxidative stress of the copper deficiency and the
[00:06:34.070]increased lipid metabolism.
[00:06:35.870]This could explain both the increased liver damage and the decreased cholesterol production.
[00:06:40.044]Additionally,
[00:06:41.630]that oxidative stress could impair cholesterol synthesis,
[00:06:44.870]which would directly lead to lipid dysregulation. Finally,
[00:06:48.500]oxidative stress could impair bile acid synthesis.
[00:06:52.700]Bile acids are the molecules that facilitate fat absorption in the intestine.
[00:06:57.560]Dysregulation of bile acids could affect overall lipid absorption from the diet,
[00:07:02.180]and again, lead to lipid dysregulation.
[00:07:04.628]Overall, this data points to the fascinating connection between copper deficiency,
[00:07:09.950]lipid metabolism, and general liver health.
[00:07:13.640]In order to more completely understand this relationship,
[00:07:16.190]further research should be conducted.
[00:07:18.110]First, bile acid levels should be tested in a variety of mouse tissues.
[00:07:22.550]Second, cholesterol and bile acid synthesis
[00:07:25.220]markers and activity levels should be tested. Finally,
[00:07:28.940]additional liver stress markers should be analyzed.
[00:07:31.970]Hopefully, the combination of these studies will lead to a more complete
[00:07:35.810]mechanistic understanding of the relationship between hepatic copper,
[00:07:39.617]liver damage, and lipid metabolism,
[00:07:42.560]and be useful in the fight against metabolic diseases.
[00:07:45.610]Finally, I would like to thank Dr. Lee, my mentor, Joe,
[00:07:48.890]and my fellow lab members for their support and advice this summer,
[00:07:52.460]I would also like to thank Dr. Becker, the SRP staff,
[00:07:56.120]the UNL Redox Biology Center, and the NSF. Thank you.

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Hepatic Copper Regulates Lipid Metabolism in High Fat Diet Fed Mice

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