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Are ceramides good anti-aging ingredients?

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Are ceramides good anti-aging ingredients?

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http://thebeautybrains.com/2015/04/are-ceramides-good-anti-aging-ingredients-episode-77/

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Do you wonder which anti-aging ingredients really work? Today we're reviewing the evidence for ceramides.
Which anti-aging ingredients really work?

When it comes to anti-aging products it's easy to be tricked into spending a lot of money on products that aren't worth it. That's because there's so much pseudoscientific misinformation out there about anti-aging cosmetic ingredients. Also, once you buy an anti-aging product, it takes you a long time to determine if it's really working for you or not. That's why we're going to focus some of our podcast episodes on specific anti-aging ingredients, Today we're talking about ceramides.

What are ceramides?

"Ceramide" is one of those buzzwords that gets thrown around a lot in the beauty industry, especially with regard to anti-aging. But I've never seen a good explanation of what a ceramide is, what it really does, and what to look for in a product. That's what we're going to cover today, starting with a little chemical background...

Ceramides are a special type of oily wax that's naturally found in our skin (and other places.) In fact, the word ceramide comes from the Latin cera which means wax. Ceramides form a kind of water-proofing barrier in the upper layers of skin. They're not only critical for helping skin retain water but they also help repair the skin's natural barrier and regulate cells. Ceramide production dwindles with age which can result in dry skin, wrinkles and even some types of dermatitis.

Did you know that newborn infants, especially premature ones, may be born with a waxy or cheese-like coating on their skin that prevents them from losing too much moisture? That coating is called the vernix caseosa and it is composed, primarily, of ceramides.

Chemically speaking, ceramides consist of a long-chain or sphingoid base linked to a fatty acid. By the way, "sphingoid bases" were first discovered in brain fluid and they're named after the Sphinx because the chemist who found thought them thought they had an "enigmatic structure." Anyway, sphingoids make up about half of a ceramide. Therefore, ceramides are not a single thing - different types of ceramides can be made depending on which specific base and which fatty acid are combined. There are at least 9 different types of ceramides found naturally. To make things even more confusing there are not only ceramides but phytoceramides, psuedoceramides, and synthetic ceramides. So let's define these before we go any further.

Ceramide: A waxy lipid that is occurs naturally in skin. It's made by combining combine a fatty acid with a sphingoid base.

Phytoceramide: A ceramide made with a phytosphingosine (a special type of sphingosine found in yeast, plants and some mammalian tissues. Don't get tricked by this because "Phyto" is a buzz word for made from plants so this sounds like a cool, green ingredient. In reality its sourced from yeast.)

Pseudo-ceramide: A lipid that has similar properties to a ceramide but which has a different structure. For example, Ceramide E is a pseudo-ceramide. Another example is Arachamide MEA. Pseudo-ceramides may be naturally occurring but typically are made synthetically.

Synthetic ceramide: A lab-created version of a ceramide found in nature.

For the most part, ceramides used in skin care are synthetic (whether they are true ceramides or pseudoceramides.) Ceramides can be sourced naturally but they are present at only low concentrations in plants and animals so naturally derived ceramides are expensive. And besides, based on what we've seen, it doesn't matter if the ceramide is natural or synthetic as long as it has the right structure.

Understanding ceramide nomenclature

Understanding which ceramides are used in cosmetics is confusing because there are three different ways they can be named:

1. The original INCI name which simply refers to each ceramide by a number.

2. The revised INCI name (sometimes called the "Motta" system) which uses a three letter designation. The first letter is the type of amide-linked fatty acid. (N stands for Normal Fatty acid. A stands for Alphahydroxy fatty acid and O stands for Omega hydroxy fatty acid.) The second letter is the type of base. (S stands for Sphinogsine base, P stands for Phytosphingosine base and H stands for Hydroxysphingosine base.) If there's an "E" in front of the two letters then that means it's an ester linked fatty acid.

3. Some times the chemical name of the ceramide is used (which doesn't include the word ceramide at all.)

What to look for on the label:

Ceramide 1 = Ceramide EOS

Ceramide 2 = Cermamide NS = N-stearoyl sphinganine

Ceramide 3 = Ceramide NP = N-stearoyl phytosphingosine

Ceramide 4 = Ceramide EOH

Ceramide 5 = Ceramide AS

Ceramide 6 = Ceramide AP = α-hydroxy-N-stearoylphytosphingosine

Ceramide 6 II = Caproyl sphingosine

Ceramide 7 = Ceramide AH

Ceramide 8 = Ceramide NH

Ceramide 9 = Ceramide EOP

Ceramide E = Cetyl-PG Hydroxyethyl Palmitamide and Hexadecanamide

Now that you know what ceramides are and how to spot them on your product labels, let's talk about what these things really do for skin. Are they worth the hype?

Ingested ceramides for skin

We're going to focus our discussion on topically applied ceramides but I want to quickly touch on ingested ceramides. If you've listened to our previous anti-aging spotlights on collagen and hyaluronic acid you know we looked at the data for ingesting those materials to help your skin. For ceramides there is SMALL amount of research that shows they can improve the skin barrier when swallowed. A company called Hitex that makes phytoceramide capsules conducted their own study that showed a "perceived" improvement in dry skin. Another study showed that taking 20mg or 40mg/daily for 3 weeks decreased transepidermal water loss (TEWL) and increased skin moisture content compared to a placebo. And, for what it's worth, the FDA has published a paper which essentially says phytoceramides are safe to ingest and that they've never seen any problems from dietary supplements that contain them. That, however, doesn't mean they've actually been proven to work. "New Dietary Ingredient Notification: For Phyto-Derived Ceramides." There just doesn't seem to be as much as a push for ingestible ceramides like we've seen with collagen.

Ceramides as topical moisturizers

Overall, topical application is much better studied and that's where the majority of interest is in the beauty biz so let's get to that.

As always we'll be using the 3 Kligman questions as a framework: is there a scientific mechanism to explain HOW ceramides work? Do ceramides penetrate into the skin where they COULD work? And are there any legitimate studies on real people showing ceramides DO work?

Is there a mechanism?
It's well understood that natural ceramides waterproof skin. Furthermore, we know they do this best when they're combined with other oily materials in a specific ratio. The optimal mixture of 50% ceramides, 25% cholesterol, and 15% free fatty acids forms what are called "crystalline lamellar structures" which have unique moisture retaining properties. So yes, there is a mechanism for how ceramides benefit skin.

Do they penetrate?
Yes they do and it's not surprising given that ceramides are "skin identical" lipids. This is not some foreign ingredient, it's one that's naturally present in the upper layers of skin. It's been proven that topically applied ceramides can move into the upper layers of the stratum corneum by a method called tape stripping. We've talked about this method before - essentially it involves sticking a piece of tape on your skin, ripping it off, and then analyzing it for the ingredient that you're looking for. Each time you do this you tear off a few more layers of skin cells so by repeated tape stripping you can get a sense of how far an ingredient penetrates into the stratum corneum. Here are two quick examples:

Friend of the Brains Dr. Zoe Draelos published one such study. Cosmetics and Dermatologic Problems and Solutions, Third Edition By Zoe Diana Draelos. Another source confirms that finding but, interestingly, the degree of penetration may depend on what else is in the formula. The Textbook of Cosmetic Dermatology says that without a glyceryl ether the ceramides weren't any better than the placebo.

Are there studies proving they work?
There a numerous studies on the efficacy of ceramide creams but there are two problems to watch out for. First, a number of the studies are "open label" which means they're not blinded and there's no control. So even if they show that ceramide cream does work you can't tell if the cream without the ceramides would have worked just as well! The second problem is that there are so many different types of ceramides, that can be used at different levels, in combination with so many other materials that's it's impossible to pinpoint a definitive study showing what works "best." Despite these problems, though, the weight of the evidence makes it apparent that ceramides can be beneficial. We'll cite a few example studies to give you a flavor of the work that's been done.

A study published in the J Clin Exp Dermatol shows that topical ceramides not only repair the skin barrier but they actually protect it from future attack by surfactants. (This study was done on mice.)

A Japanese study shows that plant-derived ceramides improve skin moisture better than a placebo.

The Kao Corporation published a study showing that a cream containing 8% of Ceramide E improves water content of skin and symptoms of atopic dermatitis. But, ceramide cream wasn't compared to any other product. So the test had no control and it wasn't blinded. By the way, this 8% concentration shows up in a couple of studies and it's MUCH higher than the typical use level of ceramides which is a few tenths of a percent.

According to the Textbook of Cosmetic Dermatology, certain ceramide combinations are better than a placebo at repairing skin barrier function.

And a paper titled "Skin-identical lipids versus petrolatum" shows that ceramides work but they aren't any better than petrolatum. They tested a blend of ceramide-3, cholesterol, oleic acid and palmitic acid and they say the lack of superiority may be due to a "suboptimal lipid mixture." Again, it's this notion that you have to have the right blend at the right ratio for ceramides to perform their best.

There are many more of these studies so it appears there is ample evidence that ceramides really do work.

Let me very quickly interject a note about a completely different approach. Instead of restoring ceramides you've lost, you can protect the ceramides you already have. There are enzymes in your skin called ceramid-ases that break down these lipids so if you can limit these enzymes theoretically you can keep more ceramides in your skin. I found one research paper on this topic and apparently it's a little bit tricky because of the difficulty in sourcing these enzymes. Researchers can't get them out of skin very easily so instead they get them from...get this...fecal extracts and nasal secretions.

So, anyway, now that we know ceramides really work what does this all mean if you want to buy an anti-aging ceramide cream?

How to pick the ceramide cream that's right for you

First, let me summarize why picking a ceramide cream is so complicated:

1. There are many different types of ceramides. But at least most of them (at least the ones commonly used) appear to be beneficial to skin.

2. Sometimes they're beneficial because they are just providing an occlusive layer on the surface of skin that locks in moisture. If that's the case, ceramides may work no better than conventional, less expensive ingredients like petrolatum.

3. Other times they're MORE beneficial because they're penetrating and moisturizing from within. This means they may have a more prolonged effect compared to conventional ingredients. However, this seems to be the case only when the ceramides are combined with other materials like cholesterol and fatty acids. AND, they have to be combined in very specific ratios. For example, in skin the natural ratio is 3.6 to 1.2 to 1. We found one patented product that uses a ratio of 3:1:1. And who know what ratios other products use - but we do know it's critical. Unfortunately we could find no side by side studies to prove which products are best. Which means that it's very difficult for you to know if any given product is worth trying, especially if it's expensive.

So, if you want add ceramides to your anti-aging regimen, here's what we recommend: Start cheap and work your way up. To help you get started, we'll list a few products starting with the inexpensive ones that may only have a single ceramide followed by more costly ones that appear to contain the optimal blend of actives (hopefully at the right ratio.) Try the cheapest one first. If you don't like the way that one makes your skin feel, go up to the next most expensive one and continue the process until you find one you like.

Product examples
Curel Ultra Healing
Cost: $0.45/oz

Comments: You can't beat the price but this Curel product only contains a single ceramide without the other critical ingredients.

Key ingredients: CETYL-PG HYDROXYETHYL PALMITAMIDE

Full Ingredient list: WATER, GLYCERIN, PETROLATUM, CETEARYL ALCOHOL, DIMETHICONE, BEHENTRIMONIUM CHLORIDE, ISOPROPYL PALMITATE, CETYL-PG HYDROXYETHYL PALMITAMIDE, BUTYLENE GLYCOL, METHYLPARABEN, BENZALKONIUM CHLORIDE, ETHYLPARABEN, CITRUS AURANTIUM DULCIS (ORANGE) PEEL OIL, AVENA SATIVA (OAT) MEAL EXTRACT, BUTYROSPERMUM PARKII (SHEA) BUTTER, EUCALYPTUS GLOBULUS LEAF EXTRACT, ACACIA SENEGAL GUM, GELATIN,.
CeraVe Moisturizing Lotion
Cost: $0.92/oz

Comments: Impossible to tell for sure without seeing the formula but this one seems to offer the best blend of ingredients at the best price.

Key ingredients: Ceramide 3, Ceramide 6 II, Ceramide 1, Cholesterol, Phytosphingosine

Full ingredient list: Water (Purified), Glycerin, Capric/Caprylic Stearic Triglyceride, Behentrimonium Methylsulfate/Cetearyl Alcohol, Ceteareth 20, Ceramide 3, Ceramide 6 II, Ceramide 1, Hyaluronic Acid, Cholesterol, Dimethicone, Polysorbate 20, Polyglyceryl 3 Diisostearate, Potassium Phosphate, Dipotassium Phosphate, Sodium Lauroyl Lactylate, Cetearyl Alcohol, Disodium EDTA, Phytosphingosine, Methylparaben, Propylparaben, Carbomer, Xanthan Gum
Triceram
Cost: $8.80/oz

Comments: An over the counter medication used to treat eczema.

Key ingredients: Strangely the ingredient list just says "ceramide" without specifying which one. It also contains linoleic acid which is good but none of the other key actives.

Full ingredient list: Active: Purified Water, Lanolin, PEG-20 Methyl Glucose Sesquistearate, Cetyl Alcohol, Ceramide, Glycerine, Petrolatum, Dimethicone, Curcumin, Soybean Sterol, Linoleic Acid, Tocopheryl Acetate, Stearic Acid, Hyaluronic Acid, Carnosine, Carbomer, Tromethamine, Propylene Glycol, Diazolidinyl Urea, Methylparaben, Propylparaben.
DHC Ceramide Cream
Cost: $27.14/oz

Comments: Even though this is called a ceramide cream it doesn't appear to contain any actual ceramides. Go figure.

Key ingredients: cholesteryl hydroxystearate, sphingolipids,

Full ingredient list: water/aqua/eau, dipropylene glycol, caprylic/capric triglyceride, squalane, stearic acid, olea europaea (olive) fruit oil, lanolin, glyceryl stearate SE, pentylene glycol, sodium PCA, methyl gluceth-10, hydrogenated lecithin, batyl alcohol, cholesteryl hydroxystearate, phenoxyethanol, behenyl alcohol, dimethicone, tocopherol, serine, butylene glycol, potassium hydroxide, allantoin, sodium citrate, pyrus cydonia seed extract, dipotassium glycyrrhizate, phospholipids, ascorbyl tetraisopalmitate, sodium hyaluronate, hydrolyzed rye phytoplacenta extract, sphingolipids, glycine soja (soybean) seed extract
Elizabeth Arden Ceramide Lift and Firm Night Cream
Cost: $42.35/oz

Comments: This product appears to have all the right pieces but who knows if the ratio is correct. However, it'll cost you!

Key ingredients: Ceramide 1, Ceramide 3, Ceramide 6 II, Linoleic Acid, Linolenic Acid, Phytosphingosine, Cholesterol, Oleic Acid,

Full ingredient list: Water/aqua/eau, Dimethicone, Butylene Glycol, Butyrospermum Parkii (shea butter), Glycerin, Cetearyl Alcohol, Isostearyl Alcohol, Caprylic/capric Triglyceride, Theobroma Cacao (cocoa) Seed Butter, Ceteth-20 Phosphate, Butylene Glycol Cocoate, Isodecyl Salicylate, Ceramide 1, Ceramide 3, Ceramide 6 Ii, Calluna Vulgaris Extract, Chondrus Crispus (carrageenan), Dioscorea Villosa (wild yam) Root Extract, Glycine Soja (soybean) Sterols, Hibiscus Abelmoschuss Seed Extract, Trifolium Pratense (clover) Flower Extract, Sodium Hyaluronate, Linoleic Acid, Linolenic Acid, Ascorbyl Palmitate, Retinyl Palmitate, Tetrahexyldecyl Ascorbate, Tocopherol, Tocopheryl Acetate, Erythritol, Glycine Soja (soybean) Oil, Caprylyl Glycol, Isohexadecane, Macadamia Ternifolia Seed Oil, Peg-40 Hydrogenated Castor Oil, Acetyl Octapeptide-3, Sodium Pca, Trehalose, Urea, Homarine Hcl, Hydrogenated Lecithin, Hydrolyzed Potato Protein, Hydrolyzed Soy Protein, Hydrolyzed Yeast Protein, Lauryl Peg-9 Polydimethylsiloxyethyl Dimethicone, Lecithin, Phospholipids, Phytosphingosine, Ammonium Acryloyldimethyltaurate/beheneth-25 Methacrylate Crosspolymer, Ammonium Acryloyldimethyltaurate/vp Copolymer, Ceteth-20, Cholesterol, Oleic Acid, Oleyl Alcohol, Peg-100 Stearate, Sodium Lauroyl Lactylate, Dicetyl Phosphate, Hydroxyethyl Acrylate/sodium Acryloyldimethyl Taurate Copolymer, Polysorbate 60, Polysorbate 80, Polyquaternium-51, Ethylcellulose, Beta-glucan, Hexylene Glycol, Xanthan Gum, Sodium Hydroxide, Thioctic Acid, Ubiquinone, Disodium Edta, Cyclohexasiloxane, Cyclopentasiloxane, Parfum/fragrance, Benzyl Salicylate, Butylphenyl Methylpropional, Hexyl Cinnamal, Hydroxycitronellal, Hydroxyisohexyl 3-cyclohexene Carboxaldehyde, Limonene, Linalool, Isopropylbenzyl Salicylate, Benzoic Acid, Methylparaben, Phenoxyethanol, Potassium Sorbate, Sodium Benzoate, Sorbic Acid, Triacetin, Chlorphenesin.
Epiceram
I don't have the price for this one since it's a prescription drug but it is approved for treating atopic dermatitis. We also know that it contains ceramide, linoleic acid, and cholesterol in the ratio of 3:1:1. It looks promising but you'll have to ask your doctor for it. http://www.epiceram-us.com/physician.html

The Beauty Brains bottom line

"Ceramides" refers to a class of ingredients which are waxy lipids naturally found in skin.

Ceramides are good moisturizers but may be not better than regular lotions unless correctly formulated.

The best formulas blend ceramides with cholesterol and fatty acids to replicate skin's natural moisture barrier.

To save money, start with the least expensive ceramide creams and work your way up until you find one you like.

Improbable Products

We also played the Improbable Products game in this week's show. Can you guess which of these three products is just made up? (Listen to the show for the answer.)

1. Robo manicurist
If you like the look of well manicured nails but you're too lazy to do it yourself, you're in luck! A Japanese company has invented a robot designed to paint your nails.

2. Smart sock
Anyone who's suffered from calluses will love this new salicylic acid impregnated sock that treats your feet while you walk.

3. Moisture monitor
Researchers have developed a sensor that detects when your skin is dry and alerts you to apply more lotion.

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Are ceramides good anti-aging ingredients? Episode 77

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Podcast: Play in new window | Download Subscribe: Apple Podcasts | Android | RSS Do you wonder which anti-aging ingredients really work? Today we're reviewing the evidence for ceramides. When it comes to anti-aging products it's easy to be tricked into spending a lot of money on products that aren't worth it.

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Comparison Review of 5 Japanese Ceramide Toners

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Comparison Review of 5 Japanese Ceramide Toners

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I've been trashing a lot of toners (among other things) from my skincare stash. I hate being wasteful but I finally came to the realization that I don't have the mental energy to force myself to use up products that don't bring me joy if I want to do other unenjoyable, adult things like eating vegetables or going to the gym, so it had to be done.

What I learned from this purge is while botanical extracts and fancy hanbang ingredients make me buy buy buy, none of those things entice me to actually use a toner. The toners I reach for day after day tend to be fragrance free and to have a targeted function. At the moment, I've been relying heavily on various ceramide toners. As all 5 in my stash survived my latest purge, so I figure I would review them.

About Ceramides, the Importance of Formulation, & My Experience with Ceramide Toners

Ceramides are waxy lipids that naturally occur in cell membranes and are a main component of the stratum corneum. In skincare, they can do some pretty amazing barrier repairing things when formulated in a 3:1:1:1 ratio with cholesterol, linoleic acid, and palmitic acid. To my knowledge, none of these toners have the proper ingredients in this ratio, so if you're looking for an intensive, reparative treatment, you might want to go elsewhere. However, after using some of these toners regularly in in heavy doses for the past couple of months, my dry and reactive skin does seem to be less sensitive, more resilient, and overall better functioning.

This category of products seems to have specific benefits to my skin I have not observed from other types of products. Specifically, when I use ceramide toners in heavy dosages, I notice:

1. My skin handles retinoids A LOT better. My Curology prescription (tretinoin + azelaic acid + zinc pyrithione) is awesome for combatting acne but causes chronic inflammation and flaking of skin around the nose and mouth. Soon after I started heavily utilizing the Matsuyama Hydrating Toner (light version), the way Curology affected my skin suddenly changed. The area around my nose is CALMER after using Curology, which blew my mind. I've known azelaic acid is supposed to be inflammation/redness fighting, but it's just not something I've ever witnessed in the previous 16+ months I've been using a tretinoin + azelaic prescription. My theory is this toner is allowing the calming effects of azelaic acid to finally supersede the irritating effects of tretinoin. I've been able to maintain this effect with the Mebika toner and regular version of the Matsuyama toners, but not with the Biyougeneki toner or Cezanne.

2. My skin regulates turnover better. I struggle with exfoliants - both physical and chemical. If I use them too regularly, my skin becomes exceedingly sensitive and irritable. If I don't use it enough, my skin tone looks dull. Two + years into being a serious skincare user (whatever that means) and I still struggle to figure out the correct balance. I've noticed that after using these toners regularly, my skin is much less prone to dullness. I haven't touched any AHAs, BHAs, or PHAs in 6 weeks, relying solely on retinoids and L-Ascorbic Acid to regulate my skin turnover, and texturally, my adult skin has never looked or felt better. I continue to use an AHA body lotion on my arms and legs (it's necessary to keep my reptile-dry skin and mild keratosis pilaris in check) but when paired with the Cezanne or Mebika toner, I've been able to reduce the frequency with which I have to use acid. My skin stays notably hydrated and soft for longer between AHA uses.

I feel the need to reiterate that these toners do not (to my knowledge) contain the proper ratio of cholesterol, linoleic acid, and palmitic acid that are necessary for barrier repair to happen so I really don't know how to explain the mechanisms by which these effects are working. That said, I have dry skin which means my skin lacks lipids, so it's possible ceramides in a watery delivery system are exceptionally good at combatting dryness. After all, ceramides are lipids and fundamentally function as an emollient, meaning they replace the absent natural skin lipids in the space between corneocytes in the [stratum corneum]. Additional benefits include the smoothing of roughtened skin thereby changing the skin's appearance and providing occlusion to attenuate TEWL and enhance moisturization.

In either case, ceramide toners work well for my skin. Everyone's skin is different so they might not work for you, but maybe they will.

Now onto the individual reviews:

Product Name
松山油脂 肌をうるおす 保湿浸透水 ライトタイプ // Matsuyama Skin Moisturizing Water, Light Type
- and -
松山油脂 肌をうるおす 保湿浸透水 // Matsuyama Skin Moisturizing Water (Regular)

Where does it fit in your routine?
I use this after cleansing and after applying first essence.

Size and Price
The regular and light bottles are identical and both contain 120ml of product. I purchased the light version from Chomotto for ¥1,404 (~$11.90 USD) through Tenso, which is a forwarding service. That was before Amazon Japan offered international shipping. It is also available on Amazon US, and Amazon Japan. I purchased the regular version from Amazon Japan for ¥1,404. It is also available on Amazon US.

Packaging
Hefty, minimalistic glass bottle. Product dispensed through a hole in the opening.

Fragrance
Blissfully fragrance free.

Color and texture
The light toner has a texture very similar to water. The regular toner is still very fluid but is more viscous and has more slip.
Ingredients
The ingredients are very similar for both toners, with the key difference being the light version has trehalose higher in the ingredient list and also contains Rice Bran Extract and Sericin, which the regular version does not contain.

Full Ingredients (Light):
水、BG、グリセリン、1,2-ヘキサンジオール、トレハロース、ダイズ種子エキス、ダイズ発酵エキス、ダイズステロール、セラミド1、セラミド2、セラミド3、セラミド5、セラミド6II、ヒアルロン酸Na、コメヌカエキス、セリシン、水添レシチン、キサンタンガム、クエン酸、クエン酸Na
Water, Butylene Glycol, Glycerin, 1,2-Hexanediol, Trehalose, Glycine Soja (Soybean) Seed Extract, Lactobacillus / Soybean Ferment Extract, Glycine Soja (Soybean) Sterols, Ceramide 1, Ceramide 2, Ceramide 3, Ceramide 5, Ceramide 6II, Sodium Hyaluronate, Oryza Sativa (Rice) Bran Extract, Sericin, Hydrogenated Lecithin, Xanthan Gum, Citric Acid, Sodium Citrate
Full Ingredients (Regular):
水、BG、グリセリン、1,2-ヘキサンジオール、ダイズ種子エキス、ダイズ発酵エキス、加水分解ダイズタンパク、ダイズステロール 、セラミド1、セラミド2、セラミド3、セラミド5、セラミド6II、ヒアルロン酸Na、トレハロース、水添レシチン、キサンタンガム、クエン酸、クエン酸Na
Water, Butylene Glycol, Glycerin, 1,2-Hexanediol, Glycine Soja (Soybean) Seed Extract, Lactobacillus / Soybean Ferment Extract, Glycine Soja (Soybean) Sterols, Ceramide 1, Ceramide 2, Ceramide 3, Ceramide 5, Ceramide 6II, Sodium Hyaluronate, Trehalose, Hydrogenated Lecithin, Xanthan Gum, Citric Acid, Sodium Citrate

Cosdna identifies butylene glycol (1) as an acne trigger in both the light lotion and regular lotion.

Performance

I've tried 4 products from Matsuyama so far (these two toners, cleansing gel, and emulsion) and although the products aren't super exciting, every single product has been a joy to use. Matsuyama is the perfect white T-shirt of skincare and I'm totally obsessed with the brand.

When my skin does get irritated, it tends to overreact. I have an arsenal of calming/anti-inflammatory products to deal with this sensitivity. Since I started using these toners regularly, I rarely find myself reaching for those anti-redness products because I haven't needed them.

Both of these toners get a good amount of use in my routine. The normal version is more hydrating than the light version and I think it's an excellent toner, but I freaking LOVE the light version. The thinner consistency of the light version allows me to apply a lot more toner, which is key for it to do its best work.

You know how foundation looks best on good skin days? A few days after I started using the light version of this toner, I started having good foundation days EVERY SINGLE DAY. I would look in the mirror and be shocked at how even and smooth my base makeup looked. There were a few moments where I would look at myself in the mirror and be really confused because it didn't look like I had foundation on but my skin also looked like it had been put through a selfie app. This toner makes my skin smoother, creating the perfect canvas for makeup. It doesn't shrink pores, but it makes my pores less texturally intrusive to my skin which makes them less noticeable, especially under makeup. I have no doubt that if I used the regular version in the same quantities as I do the light version, I would see a similar effect, but the thicker texture of the regular version makes that impractical.

Overall Rating
Matsuyama Skin Moisturizing Water Light: 5/5 Stars
Matsuyama Skin Moisturizing Water Regular: 4/5 Stars

Product Name
美容原液　ＫＴローション // Biyougen'eki Rice Ceramide & Soy Ferment Lotion

Where does it fit in your routine?
I use this after cleansing and after applying first essence.

Size and Price
This lotion contains 185ml of product. In Japan, it is dirt cheap but unfortunately super hard to source to the US. I bought it from Rakuten for ¥ 413 (<$4 USD) through a Tenso, a forwarding service.

Packaging
Plastic bottle with an orange label that dispenses lotion through a hole in the top. If you're familiar with the Hada Labo lotion products, it's the same bottle.

Color and texture
Translucent and fluid but not watery texture. It's kind of soupy? Not at all sticky.
Ingredients

Ceramides Contained in toners: Rice Bran Sphingoglycolipid, Glucosyl Ceramide

Other Noteworthy Ingredients: Soymilk Ferment Filtrate, Soybean Seed Extract, Rice Ferment Filtrate

Full Ingredients:
水、グリセリン、BG、豆乳発酵液、ダイズエキス、加水分解ダイズタンパク、コメヌカスフィンゴ糖脂質、グルコシルセラミド、コメ発酵液、カルボマー、フェノキシエタノール、カラギーナン、デキストリン、水酸化K、メチルイソチアゾリノン、水添レシチン、リゾレシチン、(スチレン/アクリル酸アルキル)コポリマーアンモニウム
Water, Glycerin, Butylene Glycol, Soymilk Ferment Filtrate, Glycine Soja Seed Extract, Hydrolyzed Soy Protein, Rice Bran Sphingoglycolipid, Glucosyl Ceramide, Rice Ferment Filtrate, Carbomer, Phenoxyethanol, Carrageenan, Dextrin, Potassium Hydroxide, Methylisothiazolinone, Hydrogenated Lecithin, Lysolecithin, Ammonium styrene/
acrylates copolymer

Cosdna identifies butylene glycol (1) and carrageenan (5) as acne triggers and carbomer (1) and carrageenan (2) as potential irritants. This lotion also uses methylisothiazolineone (MIT) as a preservative. I am not personally sensitive to MIT but it is a super common allergen so proceed with caution and please consult a medical professional if you experience any adverse effects.

Performance

This lotion is kind of the odd one out in this review. Despite the ceramide content and other nice ingredients, it doesn't do much for me beyond basic hydration. If I apply multiple layers, it does make my skin supremely soft to the touch which is really nice on days I need a quick skin texture boost but the effects are relatively short lived.
Overall Rating: 2.9/5 Stars
Product Name
セザンヌ スキンコンディショナー高保湿 // Cezanne Skin Conditioner High Moist

Where does it fit in your routine?
I use this after cleansing and after applying first essence.

Size and Price
This bottle contains 500ml of product and costs ￥702 before shipping on Amazon JP where I purchased it. It is also available on Amazon US and Ebay.

Packaging
Transparent plastic bottle. The cap and all label information is in pink.

Fragrance
None detected.

Color and texture
This lotion has a translucent white color. It is very fluid but is a tad milkier than water in both appearance and feel.
Ingredients

Ceramides Contained in toners: Ceramide AP, Ceramide NP, Ceramide EOP, Phytosphingosine

Other Noteworthy Ingredients: Coix Lacryma-Jobi Ma-yuen Seed Extract, 3 forms of Sodium Hyaluronate, Cholesterol, 7 Amino Acids

Full Ingredients:
水、グリセリン、BG、ハトムギ種子エキス、セラミドAP、セラミドNP、セラミドEOP、フィトスフィンゴシン、ヒアルロン酸Na、アセチルヒアルロン酸Na、加水分解ヒアルロン酸、サクシノイルアテロコラーゲン、ダイズ種子エキス、グルタミン酸、アロエベラ液汁、レモン果実エキス、ローズマリー葉エキス、ホップ花エキス、セイヨウアカマツ球果エキス、スギナエキス、PCA-Na、セリン、リシン、グリシン、アラニン、アルギニン、トレオニン、プロリン、コレステロール、ベタイン、アラントイン、ソルビトール、クエン酸、クエン酸Na、EDTA-2Na、ヒドロキシエチルセルロース、キサンタンガム、ラウロイルラクチレートNa、カルボマー、(スチレン/VP)コポリマー、メチルパラベン、プロピルパラベン
Water, Glycerin, Butylene Glycol, Coix Lacryma-Jobi Ma-yuen Seed Extract, Ceramide AP, Ceramide NP, Ceramide EOP, Phytospingosine, Sodium Hyaluronate, Sodium Acetylated Hyaluronate, Hydrolyzed Hyaluronic Acid, Succinoyl Atelocollagen, Glycine Soja (Soybean) Seed Extract, Glutamic Acid, Aloe Barbadensis Leaf Juice, Citrus Limon (Lemon) Fruit Extract, Pinus Sylvestris Cone Extract, Equisetum Arvense Extract, Sodium PCA, Serine, Lysine, Glycine, Alanine, Arginine, Threonine, Proline, Cholesterol, Betaine, Allantoin, Sorbitol, Citric Acid, Sodium Citrate, Sodium EDTA, Hydroxyethylcellulose, Xanthan Gum, Sodium Lauroyl Lactylate, Carbomer, Styrene/VP Copolymer, Methylparaben, Propylparaben

Cosdna identifies butylene glycol (1) as an acne trigger and carbomer (1) as a potential irritant.

Performance

After using this for 3 days on my face, it started breaking me out so now I use solely as a body product. This makes scoring difficult because everyone's acne triggers are different and I really didn't get to observe its effects on my face long enough to make any sort of informed statements about it, so I'm going to focus on what it does when I apply to my arms and legs. In general, I find this to be the heftiest and most moisturizing lotion of this bunch, which is both good and bad. It not only keeps my skin hydrated and soft but seems to make any other body products I use work better. On the other hand, I find it to be a little sticky immediately after application. It's a minor annoyance, but one that is enough to deter me from purchasing again, especially as the Mebika lotion works similarily but without the stickiness.
Overall Rating: 3.2/5 Stars
Product Name
米美糀 モイストローション // Mebika Moisture Lotion

Where does it fit in your routine?
I use this after cleansing and after applying first essence.

Size and Price
This bottle contains 500ml of product and costs ￥972 before shipping on Amazon JP where I purchased it.

Packaging
Transparent plastic bottle that is rounded on top.

Fragrance
This is 97.5% fragrance free. If I really stick my nose into it it kind of smells like rice water.

Color and texture
Translucent white, milky color and texture.
Ingredients

Ceramides Contained in toners: Ceramide 5, Rice Bran Sphingoglycolipid, Ceramide 1, Ceramide 2, Ceramide 3, Ceramide 6II

Other Noteworthy Ingredients: Rice Bran in (extract, powder, ferment filtrate, oil, protein), a ton of Extracts (ie Royal Jelly, Coix Lacryma-Jobi Ma-yuen Seed, Soybean, Artichoke, Licorice Root etc), Evening Primrose, Soy Sterol

Full Ingredients:
水、ＤＰＧ、セラミド５、乳酸桿菌／コメヌカ発酵液、コメ胚芽油、加水分解コメタンパク、コメヌカスフィンゴ糖脂質、コメ粉、コメヌカ油、コメ発酵液、コメヌカエキス、イノシトール、プラセンタエキス、水溶性コラーゲン、セラミド１、セラミド２、セラミド３、セラミド６Ⅱ、ローヤルゼリーエキス、月見草油、ソメイヨシノ葉エキス、ハトムギ種子エキス、ダイズステロール、ダイズ種子エキス、ヨモギ葉エキス、ヒマワリ種子エキス、アーチチョーク葉エキス、アマチャヅル葉エキス、ワイルドタイムエキス、ゲットウ葉エキス、ローズマリー葉エキス、マドンナリリー根エキス、オタネニンジン根エキス、カンゾウ根エキス、オウゴン根エキス、グリセリン、ＰＥＧ／ＰＰＧ／ポリブチレングリコール－８／５／３グリセリン、エチルヘキシルグリセリン、トレハロース、（スチレン／アクリレーツ）コポリマー、ヒドロキシプロピルシクロデキストリン、クエン酸Ｎａ、クエン酸、水添レシチン、リゾレシチン、キサンタンガム、ポリアクリレートクロスポリマー-１１、ＰＥＧ－６０水添ヒマシ油、ｔ－ブタノール、ＢＧ、トコフェロール、フェノキシエタノール、ブチルカルバミン酸ヨウ化プロピニル
Water, Dipropylene Glycol, Ceramide 5, Lactobacillus/Rice Bran Ferment Filtrate, Oryza Sativa (Rice) Germ Oil, Hydrolyzed Rice Protein, Rice Bran Sphingoglycolipid, Oryza Sativa (Rice) Powder, Oryza Sativa (Rice) Bran Oil, Rice Ferment Filtrate (Sake), Oryza Sativa (Rice) Bran Extract, Inositol, Placental Protein, Soluble Collagen, Ceramide 1, Ceramide 2, Ceramide 3, Ceramide 6II, Royal Jelly Extract, Oenothera Biennis (Evening Primrose) Oil, Pronus Yedoensis Leaf Extract, Coix Lacryma-Jobi Ma-yuen Seed Extract, Glycine Soja (Soybean) Sterols, Glycine Soja (Soybean) Seed Extract, Artemisia Princeps Leaf Extract, Helianthus Annuus (Sunflower) Seed Extract, Cynara Scolymus (Artichoke) Leaf Extract, Gynostemma Pentaphyllum Leaf Extract, Hymus Serpyllum Extract, Alpinia Speciosa Leaf Extract, Rosmarinus Officinalis (Rosemary) Leaf Extract, Lilium Candidum Bulb Extract, Panax Ginseng Root Extract, Glycyrrhiza Glabra (Licorice) Root Extract, Scutellaria Baicalensis Root Extract, Glycerin, PEG/PPG/Polybutyleneglycol-8/5/3Glycerin, Ethylhexylglycerin, Trehalose, Styrene/Acrylates Copolymer, Hydroxypropyl Cyclodextrin, Sodium Citrate, Citric Acid, Hydrogenated Lecithin, Lysolecithin, Xanthan Gum, Polyacrylate Crosspolymer-11, PEG-60 Hydrogenated Castor Oil, tert-Butyl Alcohol, Butylene Glycol, Tocopherol, Phenoxyethanol, Iodopropynyl Butylcarbamate

Cosdna identifies evening primrose oil (2), butylene glycol (1), and tocopherol (2) as acne triggers and evening primrose oil (2) and tocopherol (2) as potential irritants.

Performance

I started using this after I had already reaped much of the benefits of the Matsuyama light lotion, so I unfortunately cannot quite confirm it initiates the same calming and skin-resilience boosting effects, but it does work equally well in maintaining those effects. When I use this regularly, my skin is less less irritable and less sensitive. Also, my skin drinks this stuff up. Even though this is thicker than the light Matsuyama skin moisturizing water, I'm still able to apply multiple layers without any residue or stickiness, which is helpful.

Since it's so cheap and there's so much of it in a bottle, I use this on both my face and my body. On my body, it imcreases overall hydration, improves skin smoothness, and boosts/prolongs the moisturizing and exfoliating effects of the AHA body lotion I use.

In addition, of all the items discussed in this post, this lotion stands out in that it also has modest but noticeable brightening effects! This is likely due to the ferments and rice ingredients it contains, which is a really nice bonus for an already awesome product!
Overall Rating: 4.2/5 Stars
Summary + Comparison of All 5 Toners

My ranking of the toners from least favorite to most favorite is:

Cezanne High Mist Skin Conditioner - This is the heftiest / most moisturizing toner in the bunch, but on my face it breaks me out and on my body it feels just a tad sticky. Functionally, I like it enough to keep it around and finish it as a body product but I don't enjoy using it and will definitely not be repurchasing

Biyougeneki Rice Ceramide & Soy Ferment Lotion - I've had no issues with this toner but it's overall kind of unremarkable. Will not repurchase

Matsuyama Skin Moisturizing Water - I really like this toner. It hydrates and allows my skin to deal with retinoids better.

Mebika Moisture Lotion - This toner is a cornicopia of skin beneficial ingredients and is the only one in this round-up that has (modest but noticeable) brightening effects. I use this as a face and body product and like that it helps my skin handle retinoids and that I can easily apply multiple layers of this without any stickiness or residue. I also like that this helps my skin handle retinoids.

Matsuyama Skin Moisturizing Water Light Version - Like the Mebika, it helps my skin process retinoids and I can saturate my skin in multiple layers of this. It also makes my base makeup look amazing, which I really, really appreciate

In short, I use all of these ceramide toners in my routine regularly but the ones I plan on repurchasing over and over again are the Matsuyama Light toner and the Mebika moisture lotion.
Disclaimer: This product was purchased by me. This post contains an affiliate links. Purchasing an item through an affiliate link does not add any cost to the customer but does help support this site's upkeep.

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Comparison Review of 5 Japanese Ceramide Toners

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I've been trashing a lot of toners (among other things) from my skincare stash. I hate being wasteful but I finally came to the realization that I don't have the mental energy to force myself to use up products that don't bring me joy if I want to do other unenjoyable, adult things like eating vegetables or going to...

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Comprehensive quantification of ceramide species in human stratum corneum

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Comprehensive quantification of ceramide species in human stratum corneum

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Topically applied ceramide accumulates in skin glyphs

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Topically applied ceramide accumulates in skin glyphs

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Abstract

Ceramides (CERs), structural components of the stratum corneum (SC), impart essential barrier properties to this thin outer layer of the epidermis. Variations in CER species within this layer have been linked to several skin diseases. A recent proliferation of CER-containing topical skin-care products warrants the elucidation of CER penetration profiles in both healthy and diseased skin. In the current study, the spatial distributions of CER concentration profiles, following topical application of two species of CER, were tracked using infrared imaging. Suspensions of single-chain perdeuterated sphingosine and phytosphingosine CER in oleic acid were applied, in separate experiments, to the surface of healthy intact ex vivo human skin using Franz diffusion cells. Following either a 24- or 48-hour incubation period at 34°C, infrared images were acquired from microtomed skin sections. Both CER species accumulated in glyph regions of the skin and penetrated into the SC, to a limited extent, only in these regions. The concentration profiles observed herein were independent of the CER species and incubation time utilized in the study. As a result, a very heterogeneous, sparse, spatial distribution of CERs in the SC was revealed. In contrast, oleic acid was found to be fairly homogeneously distributed throughout the SC and viable epidermis, albeit at lower concentrations in the latter. A more uniform, lateral distribution of CERs in the SC would likely be important for barrier efficacy or enhancement.

Keywords: stratum corneum, infrared imaging, topical delivery, oleic acid

Introduction

The composition and molecular order of stratum corneum (SC) lipids are critical in the barrier function of skin. SC lipids are composed of approximately equimolar ratios of ceramides (CERs), cholesterol, and free fatty acids as well as small amounts of cholesterol esters and cholesterol sulfate. CERs play an essential structural role in the barrier properties of the epidermis. Besides their structural role in the SC, they also serve as intracellular signaling molecules in the viable epidermis (VE) and regulate several biological processes, such as proliferation, differentiation, apoptosis, inflammation, and immune responses. 1

Variations in skin CERs have been linked to various skin conditions. Many disorders, including atopic dermatitis 2, 3 and psoriasis, 4 often show an impaired barrier function and present either a decrease in total CER content or differences in the relative amounts of chemical species of CERs. Other skin diseases have also been linked to lipid composition and structural order abnormalities, including lamellar ichthyosis, Netherton and Chanarin-Dorfman. 5

There has been a proliferation of topical skin-care products containing CERs intended for use by typical, healthy consumers. Despite limited in vitro or clinical evidence utilizing healthy human skin, CERs have been included in hundreds of commercial skin-care products claiming to increase the skin's hydration level, repair the cutaneous barrier, prevent moisture loss, and contribute to reducing dry and flaky skin. Whereas many CER supplementation experiments conducted after acute barrier disruption of the SC demonstrated recovery, 6 CER penetration, and thus its course of action, is likely to differ in healthy skin. In a recent review, the conclusion was drawn that perturbed skin barrier recovery depends on applying the appropriate composition of SC lipids. 7 It was further stated that the exogenous, physiological lipids are transported through the SC into the stratum granulosum (SG) cells. Two separate reports have appeared studying CER transport in uncompromised, ex vivo human skin. In the first, the penetration of fluorescently labeled CERs was shown to depend on acyl chain length, 8 with fluorescence of short-chain (C6) CER observed in the VE and long chain (C24) CER observed only in the outer layers of the SC. In the second study, the permeability of a deuterated CER was shown to depend on the delivery system used. 9 It is evident that additional studies are needed to probe CER penetration in healthy skin.

While endogenous skin CERs are clearly important in maintaining the skin barrier and regulating water flux through the skin, the effect of topically applied exogenous CER on the SC is unclear. Ideally, CER supplementation of skin would result in the topically applied CER diffusing throughout the SC and strengthening its barrier properties. The initial organization of SC lipids takes place at the SG-SC interface, following enzymatic hydrolysis of lipid precursors to the final SC lipid constituents, 2, 10, 11 and suggesting that it is necessary to deliver the topically applied CER to the SG. However, it is difficult to administer the lipids deep into the interface in healthy skin using conventional formulations such as ointments and creams. 12, 13 As an alternative, formulations utilizing nanoemulsions, including solid lipid nanoparticles, for transdermal and dermal delivery of actives are being actively investigated. 14- 16

A limitation in studying CER penetration into skin has been the lack of sensitive instrumentation with the ability to differentiate exogenous CER from endogenous CER. Infrared (IR) imaging is a vibrational spectroscopy-based method, which provides molecular structure information about endogenous skin components in addition to tracking the exogenous agent whose penetration is being monitored. Over the past 20 years, the development of IR microspectroscopic imaging has proven the feasibility of acquiring spatially resolved spectra from tissue including microtomed skin sections. 17- 22 Penetration of exogenous agents is conveniently tracked from the spatial distribution of their vibrational band intensities. Although IR microscopic imaging has not been previously used, to the best of our knowledge, to measure CER penetration into skin, studies demonstrating the feasibility of the experiment have indeed appeared. Gotter et al 23 investigated the heterogeneous lateral distribution of the anti-psoriasis drug dithranol in an artificial membrane. Mao et al 19 imaged the distribution of sodium dodecyl sulfate across different skin regions. A useful advantage of IR imaging was realized from the fact that Beer's law is obeyed in the IR, thereby permitting (in the example cited) determination of the sodium dodecyl sulfate concentrations in the skin. Finally, IR microspectroscopic imaging has been used to investigate the penetration of oleic acid (OA) and glycerol trioleate in ex vivo human skin. 24

The goal of the current study is to determine if topically applied CER penetrates and augments the lipids in the SC in ex vivo intact skin. We demonstrate the feasibility of mapping CER permeation in human skin with IR imaging. We imaged the penetration of topically applied deuterated CERs with a common permeation enhancer, OA. The use of single-chain perdeuterated N-palmitoyl-D- erythro-sphingosine (CER [NS]-d31) or N-palmitoyl-D- erythro-phytosphingosine (CER [NP]-d31) permits us to spectroscopically distinguish the exogenous agents from the endogenous SC lipids without affecting penetration parameters. The CERs chosen for this study have also been identified in the final report of the 2015 Cosmetic Ingredient Review Expert Panel 25 as being among the top three CERs having the most reported uses in cosmetic and personal care products. The major use of these CERs is in leave-on skin-care preparations. In the study reported here, images of CER concentration were generated after applying extinction coefficients determined from Beer's law. The results demonstrate that CER applied with OA to intact skin remains predominantly on the surface of the SC, suggesting that topical replenishment of CER may not be an effective approach to improve the barrier properties of healthy skin.

Materials and methods

Materials

Acid-chain perdeuterated N-palmitoyl-D- erythro- sphingosine (CER [NS]-d31) and acid-chain perdeuterated N-palmitoyl-D- erythro-phytosphingosine (CER [NP]-d31) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). OA and acyl chain perdeuterated oleic acid (OA-d) were purchased from Sigma-Aldrich Co (St Louis, MO, USA). Human abdominal skin from plastic surgery procedures was obtained from several dermatological offices following informed consent and ethics board approval. Samples were fast frozen with liquid nitrogen after removal of subcutaneous fat, cut into approximately 15×15 cm ² pieces, and stored at -20°C for 6 months or less.

Methods

Sample preparation

CER was mixed with OA to make suspensions at ~10-12 mg/mL of CER (approximately five times than that commonly found in commercial formulations). Skin samples of ~2×2 cm ² surface area and ~2.5 mm thickness were cut from a larger section and defrosted on filter paper with the SC side up. The SC was cleaned with cotton swabs and tape-stripped twice to remove most of the sebum.

For the CER permeability study, skin samples were mounted and secured on a Franz diffusion cell (PermeGear, Inc., Hellertown, PA, USA) with the SC facing the donor compartment. The acceptor compartment was partially filled with distilled water, and, in separate experiments, 50 µL of each suspension was applied on the skin surface (~0.64 cm ²). Samples, wrapped with Parafilm M ® to minimize water evaporation and to keep skin hydrated, were incubated at 34°C for 24 or 48 hours in separate experiments. Control experiments were conducted under the same conditions with topical application of only OA-d or proteated CER suspensions in OA-d. After incubation, the excess suspension was removed with a cotton swab, the apparatus was disassembled, the skin surface thoroughly wiped with Kimwipes™, and the center part of the skin samples was fast frozen with liquid N ₂.

IR microscopic imaging

For IR imaging, samples prepared as described in the previous subsection were stabilized using a drop of water without embedding agents at -30°C within a Bright/Hacker 5030 Microtome (Bright Instrument Co Ltd, Huntingdon, UK; Hacker Instruments Inc., Fairfield, NJ, USA). Sections (~10 µm thick) were cut perpendicular to the SC surface and placed on CaF ₂ IR windows. IR images were collected with a PerkinElmer Spotlight 300 system (PerkinElmer Inc., Waltham, MA, USA) in transmission mode with an essentially linear array (16×1) of mercury-cadmium-telluride detector elements. A 6.25×6.25 µm ² pixel size was utilized and 32 scans were acquired at a spectral resolution of 8 cm −1. Image sizes were ~500×200 µm ², corresponding to 80 pixels in the x direction and 32 pixels in the y direction. Visible micrographs were obtained with the microscope integrated into the Spotlight 300 system. A minimum of two separate sets of skin specimens were prepared, imaged, and analyzed for each CER. Typical data are presented herein.

Determination of IR extinction coefficients of CER [NP]-d31

Chain perdeuteration of CER shifts the methylene stretching frequencies to a spectral region free from interference of endogenous skin vibrations. As IR spectra of skin sections were acquired in the transmission mode, Beer's law was applicable. To obtain extinction coefficients, CER [NP]-d31 was dissolved at known concentrations in chloroform:methanol 3:1 (v/v). A fixed path length (16.4 µm) IR cell with CaF ₂ windows was used to measure the absorbance of each standard solution. IR spectra were acquired with a Nicolet 6700 spectrometer (Thermo Fisher Scientific, Waltham, MA, USA), co-adding 128 scans and using a spectral resolution of 2 cm −1.

The area of the asymmetric CD ₂ stretching (v asymCD ₂) band (obtained using ISys software [v 5.0; Malvern Instruments Ltd, Malvern, UK]) was obtained as a function of molar concentration. Linear regression was used to find the best fit to the Beer's law plots. The extinction coefficient (mol −1 dm ³ cm −1) and correlation coefficient were 7,470 and 0.999, respectively. When calculating CER [NP]-d31 concentration, we assumed that the thickness (path length) of the skin section was 10 µm. The correlation coefficient demonstrates the high precision of using the asymmetric CD ₂ stretching band areas for calculating CER [NP]-d31 concentrations in skin.

IR imaging data analysis

Vibrational microspectroscopic images were generated from IR spectral data using ISys software (v 5.0; Malvern Instruments Ltd). Image planes of spectral parameters (integrated peak area or peak height) were produced after linear baselines were applied in spectral regions of interest. Image planes of CER concentrations were generated after using the extinction coefficient to estimate concentration. Figures were generated with SigmaPlot 2000 (SPSS Inc., Chicago, IL, USA).

Results

Permeation of CER [NS]-d31

Typical single-pixel IR spectra of CER [NS]-d31 at different depths in skin are shown in with bands of interest marked. highlights the CD ₂ stretching region (2,234-2,064 cm −1) at the same depths. By integrating the area under the asymmetric CD ₂ stretching band and converting to CER [NS]-d31 concentration utilizing Beer's law, we can quantitatively track the permeation of CER [NS]-d31 in skin. A high signal-to-noise ratio is evident in the spectra. The detection limit for CER [NS]-d31 is apparent in at a depth ~12.5 µm from the skin surface (third spectrum from top) and is 3.5 mM (~1.3 wt% of SC lipid phase) where the peak area is approximately two times the noise level.

illustrates the spatial distribution and concentration of CER [NS]-d31 penetration in skin after a 24-hour incubation period. displays the visible micrographs of several skin sections whereas highlights IR images (of the same sections) of CER [NS]-d31 concentration. Each IR image is comprised of 2,560 unique IR spectra (each pixel is observable in the image) with the CER [NS]-d31 concentration determined from the CD ₂ stretching region. CER [NS]-d31 appears to be heterogeneously distributed and localized in pockets close to the skin surface. more clearly indicates the distribution of CER [NS]-d31 where regions below the detection limit are displayed in gray. Determining CER penetration into the skin is complicated by the nonplanar nature of the skin surface; glyphs and other features result in a complex topography. To more closely delineate CER penetration into the SC, as opposed to CER that remains on top of the SC, line plots of CER concentration and Amide II peak height were compared between glyph and non-glyph regions in . In the SC, Amide II band intensity predominantly arises from keratin. The surface of the skin is defined at the half-maximum Amide II peak height (~0.2 AU in the line plots). In the glyph set of plots, labeled "1" in , there is high CER concentration at ~0-10 µm from the image edge, in the region where the Amide II peak height is less than 0.2 AU. This demonstrates that there was a significant amount of CER [NS]-d31 within the glyph outside the SC that did not actually penetrate into the SC. There is some overlap, however, at ~25 µm from the image edge where the Amide II intensity indicates the surface of the SC and where CER [NS]-d31 concentration is ~0.008-0.015 M within the SC. A similar phenomenon is also observed in line plots labeled "2" in in a non-glyph region with a relatively lower concentration of CER. The line plots labeled "3" were chosen at a place where CER was not observed in , and serve as a baseline for analysis of line plots 1 and 2. In summary, CER [NS]-d31 was heterogeneously distributed on top of the skin surface with small pockets of relatively low concentration penetrating ~10-15 µm into the SC.

Since we observed CER [NS]-d31 accumulating in glyph regions at relatively high concentrations, it was of interest to further explore these regions. shows a visible image of a skin section with a deep glyph. displays the corresponding IR image of CER [NS]-d31 concentration, where a high CER concentration can be observed in the deep glyph region. Figure 3C and D focus on the CER concentration within the glyph area marked with a white box in . This concentration image has been rotated clockwise and enlarged in , while displays several line plots of CER concentration and Amide II peak height at the regions marked in 3C. The blue solid line plot indicates the CER concentration just at the surface of the SC (Amide II peak height ~0.2 AU), whereas the other line plots display the CER concentration within the top ~18µm. CER concentrations can be observed to decrease rapidly from ~20-28 mM in the top ~6 µm to approximately the detection limit of 3.5 mM at a depth of 18-20 µm.

Permeation of CER [NP]-d31

Penetration studies with CER [NP]-d31 () show similar IR concentration profiles to CER [NS]-d31 (~4-10 mM) with visible images of the same CER [NP]-d31-treated sections shown in . Results are shown for both 24- and 48-hour incubation periods. , with skin regions where CER concentration was below the detection limit masked in gray, depicts a heterogeneous spatial distribution of CER [NP]-d31 concentrations, localized in pockets on or within the SC surface and in deep glyph areas. The line plots shown in suggest that CER [NP]-d31 was heterogeneously distributed on the skin surface with small pockets at ~8 mM penetrating ~10-15 µm into SC. There was no significant difference between the 24- and 48-hour incubation times. Similar results were found comparing incubation times for CER [NS]-d31 (data not shown).

OA penetration

Two control experiments were conducted to compare the penetration and distribution of OA in skin with the observed limited CER penetration. In the first control experiment, skin samples were only treated with OA-d. In the second, a suspension of proteated CER [NS] in OA-d was topically applied. shows the visible images of control skin sections and depicts IR images of OA-d concentration and distribution by integrating the asymmetric CD ₂ stretching band. A more homogenous distribution of OA-d can be observed penetrating into the SC and VE, mainly concentrating in the SC. In contrast to the spatial distribution of both CER classes, OA-d is not concentrated in glyph regions (see glyph region in bottom of last image on right). Similar line plots of OA-d concentration and Amide II peak height are also compared in . One line plot is shown for each type of control experiment and both show a more homogenous distribution of OA-d throughout the SC and VE.

Discussion

Few previous studies have sought to quantify the transport of topically applied CER through healthy human skin. As far as we are aware, the majority of CER penetration studies have been conducted on acutely compromised skin in which recovery of SC barrier properties is monitored by transepidermal water loss (TEWL) and/or SC hydration by conductance measurements. 6, 7 For quantitative studies, tape stripping of the SC is commonly used along with high-performance liquid chromatography analysis. Increasing numbers of tapes are assumed to be removing deeper layers of skin. While subsequent tapes do in general remove deeper layers of the SC, tape stripping as a means of depth profiling is a source of significant ambiguity. 26 One particular problem is determining what happens in glyphs. In tape stripping, material in glyphs can appear on tapes that predominantly sample layers deeper in the skin. As can be seen in -, topically applied CER appears to accumulate or be limited to the glyph regions. If tape stripping were done on these sections, the CER would appear on tapes thought to be sampling deeper within the skin. IR imaging provides CER concentration information with nonambiguous lateral and depth spatial information.

Imaging penetration using fluorescently labeled active agents also yields accurate lateral and depth spatial information; however, the fluorophore may alter penetration mechanisms. In a related report, Novotný et al 8 examined the chain-length dependence of the penetration of fluo-rescently labeled CER [NS] in skin. The current results are reasonably consistent with this work for the longer chain CERs. Whereas Novotný et al reported that C6 CER penetrated to the VE and C24 resided only in the outer SC, the C16 CERs (both [NS] and [NP]) used in the study reported here were sporadically detected in the outer SC layers. In addition, although fluorescence detection is more sensitive than IR absorption, the application of Beer's law to IR images acquired in the transmission mode yields quantitative concentration values for the CERs in the SC glyph regions.

One of the goals of the current study was to demonstrate the potential of IR imaging to track the spatial distribution of the concentration of topically applied material in skin. The concentration of CERs and the pure OA enhancer were chosen to maximize CER penetration in skin. Further studies utilizing healthy skin and CERs with a variety of head groups, chain lengths, and formulations will help us evaluate the generality of these conclusions.

The current results also suggest that when OA is used as an enhancer, the topically applied CERs used herein remain predominantly within the glyphs. The recent study of Sahle et al 9 demonstrated that various microemulsions can be used to control longer chain (C18) CER [NS] penetration across the SC. In that work, the CER concentration in upper and lower SC regions was quantified via tape stripping and high-performance liquid chromatography. In addition, uniformity in CER lateral distribution in the SC would seem to be important if CER is to incorporate into and enhance the structural barrier. Although Sahle et al 9 observed a significantly higher concentration of exogenous CER in the SC, their approach, geared toward sensitivity, only delineated CER depth distribution, with no information about the lateral variations. Without an approach such as the one used here, it is difficult to tell whether barrier enhancement is indeed occurring or whether, for example, exogenous CER is accumulating in the glyphs.

Acknowledgments

This work was generously supported by Johnson & Johnson Consumer Companies, Inc.

Footnotes
Disclosure
The authors declare no conflicts of interest in this work.

References

19. Mao G, Flach CR, Mendelsohn R, Walters RM. Imaging the distribution of sodium dodecyl sulfate in skin by confocal Raman and infrared microspectroscopy. Pharm Res. 2012;29(8):2189-2201. [ PMC free article]

24. Mack Correa MC, Mao G, Saad P, Flach CR, Mendelsohn R, Walters RM. Molecular interactions of plant oil components with stratum corneum lipids correlate with clinical measures of skin barrier function. Exp Dermatol. 2014;23(1):39-44. [ PMC free article]

# data[].attributes.attachment_link.title
Topically applied ceramide accumulates in skin glyphs

# data[].attributes.attachment_link.description
Ceramides (CERs), structural components of the stratum corneum (SC), impart essential barrier properties to this thin outer layer of the epidermis. Variations in CER species within this layer have been linked to several skin diseases. A recent proliferation of CER-containing topical skin-care products warrants the elucidation of CER penetration profiles in both healthy and diseased skin.

# data[].attributes.headline
The  role  of  ceramide  composition  in  the  lipid  organisation of  the  skin  barrier

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The  role  of  ceramide  composition  in  the  lipid  organisation of  the  skin  barrier

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The role of ceramide composition in the lipid organisation of the skin barrier - ScienceDirect

# data[].attributes.attachment_link.description
The lipid lamellae in the stratum corneum (SC) play a key role in the barrier function of the skin. The major lipids are ceramides (CER), cholesterol (CHOL) and free fatty acids (FFA). In pig SC at least six subclasses of ceramides (referred to as CER 1, 2-6) are present.

# data[].attributes.headline
Basic Nanostructure of Stratum Corneum Lipid Matrices Based on Ceramides [EOS] and [AP]: A Neutron Diffraction Study

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Basic Nanostructure of Stratum Corneum Lipid Matrices Based on Ceramides [EOS] and [AP]: A Neutron Diffraction Study

# data[].attributes.attachment_link.title
Basic Nanostructure of Stratum Corneum Lipid Matrices Based on Ceramides [EOS] and [AP]: A Neutron Diffraction Study

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The goal of this study was to investigate the nanostructure of SC lipid model membranes comprising the most relevant SC lipids such as the unique-stru...

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Endurance and Resistance Training Affect High Fat Diet-Induced Increase of Ceramides, Inflammasome Expression, and Systemic Inflammation in Mice

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Endurance and Resistance Training Affect High Fat Diet-Induced Increase of Ceramides, Inflammasome Expression, and Systemic Inflammation in Mice

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Abstract

The study aimed to investigate the effects of differentiated exercise regimes on high fat-induced metabolic and inflammatory pathways. Mice were fed a standard diet (ST) or a high fat diet (HFD) and subjected to regular endurance training (ET) or resistance training (RT). After 10 weeks body weight, glucose tolerance, fatty acids (FAs), circulating ceramides, cytokines, and immunological mediators were determined. The HFD induced a significant increase in body weight and a disturbed glucose tolerance ( p < 0.05). An increase of plasma FA, ceramides, and inflammatory mediators in adipose tissue and serum was found ( p < 0.05). Both endurance and resistance training decreased body weight ( p < 0.05) and reduced serum ceramides ( p < 0.005). While RT attenuated the increase of NLRP-3 (RT) expression in adipose tissue, ET was effective in reducing TNF- α and IL-18 expression. Furthermore, ET reduced levels of MIP-1 γ, while RT decreased levels of IL-18, MIP-1 γ, Timp-1, and CD40 in serum ( p < 0.001), respectively. Although both exercise regimes improved glucose tolerance ( p < 0.001), ET was more effective than RT. These results suggest that exercise improves HFD-induced complications possibly through a reduction of ceramides, the reduction of inflammasome activation in adipose tissues, and a systemic downregulation of inflammatory cytokines.

1. Introduction

Obesity is often associated with a systemic low grade inflammation [ 1]. One of the prominent hallmarks of obesity-induced inflammation is a shift in the phenotype of macrophage and T-cell populations present in adipose tissue (AT) reflecting in a unique set of pro- and anti-inflammatory cytokines [ 2]. Free fatty acids and saturated fatty acids (SFA) are suggested to induce inflammation by activation of Toll-like receptors (TLRs) 4 and 2 in adipose tissue and macrophages, culminating in an increased activation of nuclear factor kappa-B (NF- κB) thus increasing the expression in proinflammatory cytokines like TNF- α or IL-6 [ 2, 3]. Apart from SFA, a deregulated ceramide synthesis has also been suggested to play an important role in obesity related inflammation [ 4, 5].

Aerobic exercise is known to have triglyceride-lowering effects accompanied by a reduction of visceral and abdominal adipose tissue in overweight and obese subjects [ 6, 7]. These metabolic effects of exercise training are regularly accompanied by a reduction of the inflammatory status [ 8, 9]. Thereby, exercise training was followed by a reduced expression of both TLRs and several proinflammatory cytokines [ 9, 10]. However, most studies on exercise effects on obesity investigated the effect of aerobic/endurance training [ 3, 11]. Resistance training received less attention, but there are several evidences that it also affects metabolic disorders and inflammation in obesity [ 12- 16].

Insulin resistance is a common complication of obesity-induced inflammation and the reduction of blood glucose a desirable outcome of weight loss programs [ 17]. In recent years, the nucleotide-binding domain and leucine-rich-repeat and pyrin domain containing 3 (NLRP-3) inflammasome has been identified as a major contributor to the development of systemic inflammation and insulin resistance [ 18]. NLRP-3 assembly is initiated upon TLR activation, extracellular ATP, or active interleukin 1 β (IL-1 β). Expression of proIL-1 β and proIL-18 is NF- κB dependent which in turn depends on activation through TNF- α. The maturation of NLRP-3 renders caspase 1 active that cleaves inactive proIL-1 β and proIL-18 into their active forms which are then secreted into the surrounding tissue [ 18].

It was also found that ceramides induced the NLRP-3 inflammasome, driving the secretion of IL-1 β followed by the development of insulin resistance [ 18]. Ceramides induce insulin resistance through the dephosphorylation of serine/threonine kinase Akt/PKB, hence disabling the translocation of GLUT4 to the cell membrane and reducing the uptake of glucose [ 19- 21]. Aerobic exercise reduced ceramides in humans and ceramidase activity in rat skeletal muscle [ 22]. However, the effects of differentiated exercise regimes on ceramide levels, AT inflammation, and systemic cytokine levels in mice have so far not been investigated in one cohesive study. Therefore, we investigated whether different exercise regimes affect glucose tolerance, serum fatty acids and ceramides, inflammatory parameters in AT, and several inflammatory cytokines in serum of HFD fed mice. We hypothesized that both types of exercise are effective in reducing glucose intolerance. Moreover, these effects might be achieved most likely via modifying serum ceramide levels as well as local and systemic inflammatory markers. Endurance and resistance training differ in their muscle fiber response. Whereas endurance training addresses mainly type I and type IIA muscle fibers, resistance training is followed by a hypertrophy of types II and IIX [ 23, 24]. Finally, it is suggested that endurance and resistance exercise might address different targets of these pathophysiological signaling cascades. Therefore, a differentiated effect of endurance and resistance training would be expected.

2. Material and Methods

2.1. Animals, Diets, and Experimental Design

Male C57BL/6 mice ( n = 36), 10 weeks of age, were kept at 21 ± 1°C in a reverse dark-light cycle (light on from 9 pm to 9 am). Mice were randomly assigned to 6 groups (5 to 8 mice per group): standard chow/sedentary (ST/S, Altromin 1320, Altromin Spezialfutter GmbH & Co., Germany), standard chow/endurance training (ST/ET), standard chow/resistance training (ST/RT), high fat/sedentary (HFD/S, TD 03584, Harlan Teklad, Germany), high fat/endurance training (HFD/ET), and high fat/resistance training (HFD/RT). The assigned diet was fed for 4 weeks before exercise protocols were implemented. Briefly, the ST diet contained 3.4% lipids and the HFD 35% by weight. A more detailed specification of the fatty acid composition can be found in . Mice were caged separately, with free ad libitum access to food and water. The animals were put on their diet 4 weeks before the 10-week exercise protocols commenced, which means a total of 14 weeks on the diet. Weight was recorded before and after 14 weeks on the diet. The animals were handled according to the Guide for the Care and Use of Laboratory Animals. The experimental design was approved by the Animal Welfare Officer of the Justus-Liebig-University and the regional council of the city of Giessen (number 94/2010).

Table 1

2.2. Determination of Endurance Capacity and Exercise Protocols

Before ET commenced, VO ₂max was determined for each individual animal as described previously [ 25]. Eighty percent VO ₂max was used as threshold for the following exercise protocol. Each mouse assigned to the endurance group was trained on an electrical treadmill (custom-made) at 80% VO ₂max for 30 min/day, 5 times/week for 10 weeks. Briefly, by using a treadmill spiroergometry (custom-made), maximal oxygen uptake (VO ₂max) and maximal running speed (Vmax) of mice were determined. The treadmill was placed in a metabolic chamber where air was led through at a rate of 0.5 L/min. Samples of 200 mL/min of gas were extracted to the paramagnetic oxygen analyzer (type 1155, Servomex) and the carbon dioxide analyzer (Lair 12, M&C Instrument). All animals were acclimated to the treadmill before VO ₂max and Vmax were tested during a continuous, progressive test on the treadmill ergometer until exhaustion. After 10 min of acclimatization in the treadmill chamber the test uptake started at 0.15 m/s, increasing every 3 min by 0.05 m/s. The protocol for the resistance has recently been published [ 26]. Isometric strength training was performed by the following experimental setup. The mice gripped with their front and their back paws on a horizontal wire of the metal mesh and the plate was placed in a vertical position. The orientation of the mice was holding head up. Strength training was performed for 5 times/week for 3 minutes and 3 series. Break between each series was 1 minute. It was previously demonstrated that this type of isometric strength training was followed by an increase of maximum holding time in isometric strength test, a type II fiber hypertrophy, and an increased GLUT4 protein content in the membrane fraction [ 26].

2.3. Glucose Tolerance Test (GTT)

Glucose tolerance tests were performed at the end of the 10-week experimental period as follows: after an overnight 12-hour fast, blood was drawn from the tail vein of all animals (approximately 10  μL) and glucose concentration measured (glucometer, Roche Diagnostics, Mannheim, Germany). The last training occurred three days before GTT was performed. This was designated as time point zero of the GTT. Glucose (2 g/kg) was applied to the mice via intraperitoneal injection [ 27]. Blood glucose measurements were repeated after 30, 60, and 120 min.

2.4. Tissue Samples and Sample Preparation

Mice were euthanized by 5% isoflurane anesthesia followed by cervical dislocation. The animals were in the fed state when they were euthanized. Blood was collected in EDTA-coated tubes (Sarstedt, Germany) by cardiac puncture with heparin solution-washed 27G syringes (Sarstedt, Germany) and centrifuged for 10 min at 4°C and 1200 ×g. Plasma was stored at −80°C until further analysis. AT surrounding mesenteric lymph nodes was removed immediately after euthanasia and stored at −80°C. We used real-time quantitative PCR to determine TNF- α, NLRP-3, IL-1 β, and IL-18 in isolated AT. The plasma was used for a multiplex ELISA approach (Mouse Inflammation MAP, Myriad RBM, Germany) as well as for determining circulating total fatty acids and ceramides. Serum was tested for the following cytokines: CD40 (CD40), CD40 Ligand (CD40-L), C-reactive protein (CRP), endothelin-1 (ET-1), eotaxin, epidermal growth factor mouse (EGF mouse), factor VII, fibrinogen, fibroblast growth factor 9 (FGF-9), fibroblast growth factor basic (FGF-basic), granulocyte chemotactic protein-2 mouse (GCP-2 mouse), macrophage colony-stimulating factor (M-CSF), growth-regulated alpha protein (KC/GRO), haptoglobin, immunoglobulin A (IgA), interferon gamma (IFN-gamma), interferon gamma-induced protein 10 (IP-10), interleukin- (IL-) 1 alpha (IL-1 alpha), IL-1 beta, IL-10, IL-11, IL-12 subunit p70 (IL-12p70), IL-17A, L-18, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, leukemia inhibitory factor (LIF), lymphotactin, macrophage colony-stimulating factor-1 (M-CSF-1), macrophage inflammatory protein-1 alpha (MIP-1 alpha), macrophage inflammatory protein-1 beta (MIP-1 beta), macrophage inflammatory protein-1 gamma (MIP-1 gamma), macrophage inflammatory protein-2 (MIP-2), macrophage inflammatory protein-3 beta (MIP-3 beta), macrophage-derived chemokine (MDC), matrix metalloproteinase 9 (MMP9), monocyte chemotactic protein-1 (MCP-1), monocyte chemotactic protein 3 (MCP-3), monocyte chemotactic protein-5 (MCP-5), myeloperoxidase (MPO), myoglobin, oncostatin-M (OSM), serum amyloid P-component (SAP), serum glutamic oxaloacetic transaminase (SGOT), stem cell factor (SCF), T-cell-specific protein, RANTES, thrombopoietin (TPO), tissue factor (TF), tissue inhibitor of metalloproteinase 1 mouse (Timp-1 mouse), tumor necrosis factor alpha (TNF-alpha), vascular cell adhesion molecule-1 (VCAM-1), vascular endothelial growth factor A (VEGF-A), and von Willebrand factor (vWF) by a multiplexed fluorescent bead-based immunoassay (Luminex, Myriad RBM, Austin, Texas).

2.5. RNA Isolation, cDNA Synthesis, and Quantitative Real-Time PCR of Adipose Tissue

Adipose tissue (AT) was isolated from mice (for groups see above) and immediately frozen with liquid nitrogen and stored at −80°C until use. RNA was isolated by a combination of Trifast (Peqlab, Erlangen, Germany) and RNeasy Mini Kit (Qiagen, Hilden, Germany). Respective tissue was homogenized with a Precellys 24 (Peqlab, Erlangen, Germany) homogenizer in the presence of Trifast, followed by 5 min incubation on ice and centrifugation at 4°C and 12000 ×g for 10 min. The supernatant was mixed with 200  μL chloroform, followed by 10 min incubation at room temperature. After centrifugation at full speed for 15 min, the upper phase was mixed with an equal amount of 70% ethanol. The mix was loaded on an RNeasy Mini spin column and RNA isolated according to the manufacturer's instructions. The RNA concentrations were measured with a NanoDrop ND-1000 (Kisker-Biotech, Steinfurt, Germany). The isolated RNA (500 ng, each) was converted to cDNA using the iScript cDNA Synthesis Kit (BioRad, Munich, Germany). The conditions for the reverse transcription were as follows: 1 cycle at 25°C for 5 min; 1 cycle at 42°C for 30 min; 1 cycle at 85°C for 5 min. Relative quantification of IL-1 β, TNF- α, NLRP-3, and IL-18 was performed by quantitative real-time PCR with the iQ SYBR Green Supermix in duplets according to the manufacturer's instructions (BioRad, Munich, Germany). Per reaction, a 25  μL mixture was used containing 12.5  μL iQ SYBR Green Supermix, 0.5  μL forward and reverse primer (RPL32-F: 5′-TGGAGGTGCTGCTGATGTG-3′; RPL32-R: 5′-GCGTTGGGATTGGTGACTCT-3′ (product size: 127 bp); IL-1 β-F: 5′-TGGTGTGTGACGTTCCCATT-3′; IL-1 β-: 5′-TCGTTGCTTGGTTCTCCTTG-3′ (product size: 172 bp); NLRP-3-F: 5′-TCCAGCACCCAGGCTGTAAC-3′; NLRP-3-R: 5′-TGCAGAGCAGGTGCTTCAGT-3′ (product size: 189 bp); TNF- α-F: 5′-AGGGCTGTGGGACCTAAATGT-3′; TNF- α-R: 5′-ATGGGATGAGTATGGGGCAGC-3′ (product size: 199 bp); IL-18-F: 5′-GCCGACTTCACTGTACAACCG-3′; IL-18-R: 5′-GAGGGTCACAGCCAGTCCTC-3′ (product size: 182 bp)), 9.5  μL sterile water, and 2  μL of the 1 : 5 diluted complementary DNA template. A nontemplate control was performed in each run. The conditions of the real-time PCR (Mx3000P, Stratagene, Heidelberg, Germany) were as follows: 1 cycle at 95°C for 10 min and then 40 cycles at 95°C for 10 s, 59°C for 10 s, and 72°C for 10 s, followed by a dissociation curve. The intron-spanning primers were designed by using sequence information from the NCBI database. The threshold cycle (Ct) values were normalized to the endogenous control (ribosomal protein L32, RPL32), n = 3-4. The specificity of the primer pair products was tested by melting curves and agarose (1.6%) gel electrophoresis.

2.6. RNA Isolation, cDNA Synthesis, and Quantitative Real-Time PCR of Liver Tissue

The expression of the following enzymes was investigated: ceramide synthase 2 (CerS2), ceramide synthase 4 (CerS4), fatty acid elongase 1 (ELOVL1), fatty acid elongase 3 (ELOVL3), alkaline ceramidase 2 (ACER2), serine palmitoyltransferase, long chain base subunit 2 (SPTLC2), sphingomyelin phosphodiesterase 1 acid lysosomal (SMPD1), and stearoyl-CoA desaturase (delta-9-desaturase, SCD1). RNA isolation, cDNA synthesis, and qPCR analysis were performed as described recently in detail [ 28]. In brief, total RNA was isolated from 10 mg liver aliquots using TRIzol reagent (Invitrogen, Karlsruhe, Germany), and RNA concentration and purity were estimated from the optical density at 260 and 280 nm (Infinite 200M microplate reader, Tecan, Männedorf, Switzerland). RNA integrity was assessed by confirming intact bands corresponding to the 18S and 28S ribosomal RNA subunits using 1% agarose gel electrophoresis. Following cDNA synthesis within one week after RNA isolation using dT18 primer and M-MuLV Reverse Transcriptase (MBI Fermentas, St. Leon-Rot, Germany), qPCR runs were performed with a Rotorgene 2000 system (Corbett Research, Mortlake, Australia) as described recently in detail [ 28]. The used primers were as follows: ceramide synthase 2-F: CAAAGCTGGACCAAGGTTCC, ceramide synthase 2-R: GAGAGGGAGGCAGTGAGATC, ceramide synthase 4-F: CTGTGGGTGTCCTTGTAGTCC, ceramide synthase 4-R: TCCTCTGGCTTTGGTTTCTG, fatty acid elongase 1-F: TCCCATCCCTTCTCCCAGCC, fatty acid elongase 1-R: CCAGCCCTTTCAGCCTCCAG, fatty acid elongase 3-F: AGACCTACCGGCTGTCCTCC, fatty acid elongase 3-R: GAACCGAGTGAGCGTCCAGG, alkaline ceramidase 2-F: AGCCCGCCATCAACAATA, alkaline ceramidase 2-R: CCAGCAGAAGAGAGCCAGAG, serine palmitoyltransferase, long chain base subunit 2-F: CCTGTCAGCAGCTCATACCA, serine palmitoyltransferase, long chain base subunit 2-R: CACACTGTCCTGGGAGGAAT, sphingomyelin phosphodiesterase 1, acid lysosomal-F: GCCAGTCAGCCGTCCATC, sphingomyelin phosphodiesterase 1, acid lysosomal-R: CCCAGAAGCAAGCCACAAG, stearoyl-CoA desaturase (delta-9-desaturase)-F: CGTGGCTTCTTCTTCTCTCTCA, and stearoyl-CoA desaturase (delta-9-desaturase)-R: CTTCTCGGCTTTCAGGTCAG. All primer pairs were designed to have melting temperatures of about 60°C, and if possible, both primers of a primer pair were designed to be located in different exons. Ct values of target genes and reference genes were obtained using Rotorgene Software 5.0 (Corbett Research). For determination of relative expression levels relative quantities were calculated using GeNorm normalization factor. In order to calculate the normalization factor, all Ct values were transformed into relative quantification data by using the 2 −ΔCt equation, and the highest relative quantities for each gene were set to 1. From these values the normalization factor was calculated as the geometric mean of expression data of the three most stable out of six tested potential reference genes (ATP5B, CANX, CYC1, EIF4, GAPDH, and RPS9). Reference gene stability across samples from each tissue and each cell line was determined by performing GeNorm analysis [ 29]. After normalization of gene expression data using the calculated GeNorm normalization factor, means and SD were calculated from normalized expression data for samples of the same treatment group.

2.7. Measurement of Circulating Fatty Acids and Ceramides

Lipids were quantified by direct flow injection electrospray ionization tandem mass spectrometry (ESI-MS/MS) in positive ion mode using the analytical setup and strategy described previously [ 30]. In brief, plasma samples were extracted according to the method by Bligh and Dyer [ 31] in the presence of nonnaturally occurring lipid species used as internal standards. A fragment ion of m/z 264 was applied for sphingosine-based ceramides (Cer) and hexosylceramides (HexCer) with N-heptadecanoyl-sphingosine as internal standard [ 32]. Fatty acid methyl ester was formed by methanolic acetyl-chloride derivatization and extracted with hexane. Total fatty acid (FA) analysis was carried out by gas chromatography coupled to mass spectrometry (GC-MS) [ 33].

2.8. Statistical Analysis

Two-way ANOVAs with Bonferroni post hoc testing were performed in order to analyze differences between treatments. For the analysis of FA and ceramides a Bonferroni correction was applied with significance levels of p < 0.002 (fatty acids) and p < 0.005 (ceramides). We present the proinflammatory panel measured by multiplex ELISA as a heat map. To compare the cytokine production between the groups we used a two-way ANOVA with Bonferroni correction and set the new significance cut-off at p < 0.001. In order to calculate the corrected p value we divided p < 0.005 by the number of compared parameters. Furthermore, we calculated percentage changes of the cytokine levels of the HFD groups relative to the mean of the ST/S group. For the analysis of the RT-PCR results from AT, the comparative Ct method was used. Data are shown as 2(delta)(delta) Ct (ΔΔCt) that were calculated by subtracting the mean ΔCt (Ct (reference gene) − Ct (gene of interest)) of control sample from the ΔCt of each examined sample. Two-way ANOVA was used to compare the ΔCt values between the treatments. The statistical analysis of hepatic mRNA expression was performed as a two-way ANOVA on the relative mRNA levels calculated as described above.

3. Results

3.1. Exercise Reduces Glucose Intolerance and Restricts Weight Gain in HFD Mice

Diet and exercise had significant effects on blood glucose ( p < 0.01) after glucose challenge. Compared to ST/S group blood glucose levels in the HFD/S group were significantly higher at all-time points after glucose challenge ( p < 0.001). Both types of exercise led to a significant reduction of glucose intolerance in the HFD animals when compared with the sedentary group. However, the effect was more pronounced after ET ( p < 0.001; ). Mice fed the HFD gained significantly more weight than the mice fed the ST diet ( p < 0.001, ). Within the high fat group, both ET ( p < 0.05) and RT ( p < 0.001) attenuated the weight gain when compared with the sedentary group. VO ₂max change was measured at baseline before exercise commenced as well as after 10 weeks on exercise. The percent changes in VO ₂max for each diet and exercise group are shown in . ET but not RT led to significant increases of VO ₂max in both ST and HFD groups ( p < 0.001).

3.2. Ceramide Profiles Are Affected by Exercise

The HFD significantly increased most of the total fatty acids and ceramides in plasma of mice from HFD/S group ( p < 0.005; Tables and ). 13 out of 19 fatty acids analyzed were increased while 6 were unaffected. Neither ET nor RT was able to reduce significantly any fatty acid compared to the enhanced levels of sedentary mice. Eight out of 10 ceramides analyzed increased significantly in plasma from HFD/S mice compared to ST/S mice (). ET and RT affected ceramide levels slightly different in HFD mice. While ET decreased Cer d23:0 and d24:1, RT was followed by a significant decrease of Cer d24:1 and HexCer d24:1 ( p < 0.005; ).

Table 2

Table 3

3.3. Exercise Reduces Hepatic Expression of CerS4 and SPTLC2 in HFD Mice

Endurance training significantly decreased the relative mRNA levels of CerS2, ELOVL1, and ELOVL3 as well as SMPD1 ( p < 0.05) in comparison with the sedentary ST group (Figures , , , and ). Furthermore, ET increased the relative mRNA level of CerS2 ( p < 0.05) in the HFD in comparison to the corresponding ST fed mice. In contrast, ELOVL3 was strongly reduced in the ET group due to the HFD feeding. RT had no effect on CerS2 and ELOVL1 expression. However, ELOVL3 was reduced by RT only in the ST group (Figures and ). ELOVL1 and ELOVL3 mRNA expression decreased in the sedentary group due to the high fat diet. SCD1 and ACER2 were not significantly changed in the ST groups. However, we observed a trend towards a reduction of mRNA expression due to exercise in the HFD groups that did not reach statistical significance (Figures and ). The comparison within the HFD groups revealed that ET significantly reduced CerS4 and SPTLC2 mRNA. Resistance training had a similar effect on SPTLC2 but did not reach significance for CerS4 ().

3.4. Exercise Reverses HFD-Induced Alterations of Proinflammatory Cytokines

After exposure to HFD a number of cytokines increased in sedentary animals. Plasma concentrations of eotaxin, haptoglobin, MCP-1, MCP-3, MPO, CD40, KC/GRO, SAP, VCAM-1, vWF, Timp-1, IP-10, IL-18, and MIP-1 γ were significantly increased in HFD/S group compared to the ST/S ( p < 0.001). Alterations of some cytokines could be reversed by exercise intervention. Again, exercise regimes differently affect cytokine expression in the HFD groups (). While ET significantly reduced cytokine levels of MIP-1 γ, RT was followed by a significant reduction of CD40, IL-18, MIP-1 γ, and Timp-1 ( p < 0.001). In addition, we show in , as a supplementary graph, the percentage change of selected cytokines levels after both ET and RT in the HFD groups which represent the most prominent effects. This should illustrate the effect of exercise on the cytokine level of high fat diet fed mice. Notably, mice fed the HFD showed a decrease of plasma immunoglobulin A ( p < 0.001) which was not affected by any exercise intervention.

3.5. Exercise Differently Downregulates the Expression of Inflammatory Markers in Adipose Tissue

Feeding the HFD was followed by a significant increase of IL-18, TNF- α, and NLRP-3 mRNA expression in AT, while the enhanced mRNA expression of IL-1 β did not reach significance (Figures -). The two types of exercise intervention affected the inflammatory markers differently. ET was accompanied by a significant reduced expression of TNF- α and IL-1 β compared to HFD/S group. In contrast, RT significantly reduced only the NLRP-3 expression (). A secondary finding was that both exercise treatments were effective in enhancing IL-18 expression in AT of ST fed mice.

4. Discussion

We showed that HFD-induced obesity was associated with a substantial increase in body weight, a decrease of physical performance, elevated levels of serum ceramides and fatty acids, both a local, in AT, and a systemic inflammatory response, and finally glucose intolerance. Both training interventions, endurance as well as resistance exercise, were effective in reversing or in attenuating the diet-induced glucose intolerance. However ET reduced blood glucose more efficiently than RT ( p < 0.001) probably due to higher energy expenditure in the ET group. It is speculated that the exercise effect might be mediated via metabolic and inflammatory pathways as indicated by decreased levels of serum ceramides and reduced levels of tissue and serum inflammatory markers.

Obesity-associated tissue inflammation is assumed to be an important factor in the activation of the innate immune system leading to a systemic chronic low grade inflammation. It has been suggested that the starting point for AT inflammation might be the cell stress-associated accumulation of metabolites which have been shown to dramatically increase in response to HFD feeding [ 34]. In this regard, ceramides may represent a potential upstream initiating event for obesity-induced inflammation [ 18]. Current data demonstrated that high fat feeding induced an increase of several ceramides in serum, an observation that is in line with previous studies [ 19]. Likewise, ceramide transported in LDL has been shown to be elevated in the plasma of obese patients with type 2 diabetes [ 35]. Incubating cultured myotubes with reconstituted low density lipoprotein ceramide 24:0 promoted ceramide accrual in cells and was accompanied by reduced insulin-stimulated glucose uptake. Moreover, in lean mice the infusion of LDL-ceramide reduced insulin-stimulated glucose uptake, and this was due to impaired insulin action specifically in skeletal muscle [ 35]. Ceramide seems to induce insulin resistance by inhibiting insulin signal transduction, principally at Akt [ 36]. Ceramide is also postulated to activate proinflammatory pathways in macrophages, perhaps via amplification of TLR 4-mediated inflammation [ 18]. Therefore, it has been suggested that changes in plasma long chain ceramides might link the effects of HFD on the inflammatory status of adipose tissue. We did not determine macrophage phenotype in adipose tissue but showed that an increased expression of TNF- α, IL-18, and NLRP-3 which was detected in adipose tissue of HFD/S group might indicate adipose tissue inflammation. These findings are in line with previous studies which found an increased inflammatory status in AT in response to chronic high fat feeding [ 34, 37]. Interestingly, both exercise regimes differently affected expression of inflammatory markers in AT. While ET significantly reduced the expression of TNF- α, IL-1 β, and IL-18, RT reduced NLRP-3 expression. Thereby, a reduced expression of TNF- α and IL-1 β might be interpreted as a reduction of macrophage and lymphocyte activation [ 10]. Although we did not determine caspase activity, the reduced expression of IL-1 β might represent a decrease in the maturation of IL-1 β by a decrease in NLRP-3 dependent caspase 1 activity [ 18, 38]. However, a reduction of caspase 1 activity has been shown to be linked with lower levels of ceramides in serum, which has been demonstrated in our study after the exercise interventions [ 38]. Therefore, our study suggests that a starting point for the anti-inflammatory effects of exercise training might be the reduction in ceramide levels. These results are in line with previously published research that demonstrated the essential involvement of ceramides in the development of insulin resistance [ 39, 40]. Exercise significantly decreased Cer d18:1/23:0 and d18:1/24:1 in the HFD/ET group and d18:1/24:1 and HexCer d18:1/24:1 in the HFD/RT group when compared to the HFD/S group. The ceramides measured are secreted by the liver and bound to lipoproteins. It has been shown that Cer 24:1 and 24:0 make up about 80% of all measured plasma ceramides. Ceramides like reconstituted low density lipoprotein ceramide 24:0 were found to increase TNF- α, IL-1 β, IL-6, and MCP-1 in vitro and to induce insulin resistance in lean mice [ 35]. It is therefore assumed that the exercise-induced reduction of plasma long chain ceramides is at least one reason for the reduced expression of inflammatory markers in serum and AT as well as the increased glucose tolerance.

Because of the reduced levels of ceramides in plasma, we measured whether exercise affects the expression of genes involved in the synthesis of ceramides. CerS2 and ELOVL1 have been shown to be essential for the synthesis of Cer d24:0 and Cer d24:1 [ 41]. The hepatic mRNA expression levels of CerS2 and CerS4 do not change significantly in the sedentary HFD mice groups which might suggest adequate substrate availability under condition of our HFD protocol. ELOVL1 and ELOVL3 decreased significantly in the HFD/S group in comparison with the ST/S group. Since the HFD feeding led to a significant increase in long chain FAs, this downregulation of elongases might indicate a feedback loop mechanism where a high pool of FA downregulates mRNA expression. In murine hepatocytes, the expression of CerS4 is far below that of CerS2, which is the most abundantly expressed ceramide synthase in mammals. CerS2 and CerS4 share some substrate specificity; for example, CerS4 utilizes C18-C22 FA, whereas CerS2 utilizes C20-C26 FA [ 42, 43]. Furthermore, the synthesis rate of CerS in vitro has been shown to be dose-dependent and that ceramide synthesis is governed by the availability of FA [ 44, 45]. In addition, our results showed that SPTLC2 expression was significantly reduced by exercise which might also point towards a decline of available FA for ceramide synthesis. Therefore, we can speculate that exercise affects blood ceramide profile mostly through an alteration of substrate availability for ceramide synthesis rather than a change in expression of gene involved in the ceramide pathway.

Another reason for the reduced expression of inflammatory markers in serum and AT might be the effects of both exercise and diet on the expression of IL-18. IL-18 is generally considered a proinflammatory cytokine, associated with obesity and insulin resistance. Alterations in IL-18 might have been responsible for both the downregulation of glucose tolerance in HFD/S group and the increased glucose sensitivity after exercise training. These assumptions are supported by previous studies which found that obese individuals had higher IL-18 mRNA content in the abdominal adipose tissue than nonobese subjects and that exercise training lowered the elevated IL-18 mRNA levels in obese subjects [ 46]. Activation of the NLRP-3 inflammasome in course of diet-induced obesity in mice led to an increase in IL-18 secretion and was shown to affect insulin signaling [ 18]. In addition, it has been shown that IL-18 mRNA and plasma IL-18 correlated with insulin resistance, suggesting that training-induced reduction in IL-18 expression may be a contributing mechanism to improve insulin sensitivity after training [ 18, 46, 47].

We found that IL-18 mRNA expression in AT was increased in all high fat groups but was not changed by exercise. The levels of plasma IL-18, however, were significantly increased in the HFD/S group and were reduced by endurance as well as by resistance training. This was associated with improved glucose tolerance in the exercised groups [ 46]. The results of our study therefore suggest that the reduction of NLRP-3 levels in AT was not effective enough in reducing IL-18 mRNA expression in AT. However, we speculate that it affected posttranslational modification leading to a reduced maturation of IL-18 protein in AT or a reduced mobilization of IL-18 into the blood stream. The role of IL-18 during development of obesity seems to be ambivalent as it has been shown that deficiency of either IL-18 or IL-18 receptor triggers obesity and hyperinsulinemia in mice and that treatment with recombinant IL-18 reversed these effects [ 47]. Additionally, IL-18 elevated adenosine monophosphate-activated protein kinase in skeletal muscle which is known to increase energy expenditure and endurance in mice [ 48, 49]. These studies indicate that IL-18 might play an essential role in metabolic regulation and exercise adaptation. In the current study the expression of IL-18, in AT, was significantly increased in the exercised standard fed mice when compared with the ST/S animals. Additionally, we did not detect elevated glucose levels in the exercising ST groups. These results suggest that the role of IL-18 in AT is that of a metabolic key cytokine which might also be involved in the adaptation to exercise.

In addition to IL-18, exercise was effective in reducing TNF- α mRNA expression which might represent another pathway of action. Regarding the connection between inflammation and insulin resistance, it is known that TNF- α induces insulin resistance in vitro and in mice [ 50]. However, in contrast to IL-18 a decrease of TNF- α was found in AT after exercise while no changes of peripheral TNF- α were detected.

The localized inflammatory environment inside the adipose tissue seemed to have systemic consequences as reflected by enhanced levels of several proinflammatory serum cytokines. A cluster of proinflammatory cytokines increased in the HFD/S group might indicate a state of chronic low grade inflammation [ 17]. Thereby, notable increases of CD40 might be the result of an activation of lymphocytes, while cytokines like IL-18, MIP-1 γ, and Timp-1 might point towards an increased activation of macrophages [ 8]. Both exercise types reversed the diet-induced increase of some of these cytokines. While RT mainly affected CD40, IL-18, MIP-1 γ, and Timp-1 expression, ET effectively reduced MIP-1 γ. Since both exercise types reduced body weight, we cannot exclude the possible confounding effect of this weight loss and potential lower body fat content on the reduction of inflammatory markers. A major limitation of this study is that our HFD model contained 34% lipids by weight, which is a moderate challenge. However, we propose that exercise diminishes both lymphocyte and macrophage activity which is in accordance with previously published data [ 10].

Taken together, we show that HFD-induced obesity is accompanied by an increase of ceramides, activation of inflammasome in AT, and a secretion of several proinflammatory cytokines including IL-18 that all are known to affect glucose sensitivity. Both ET and RT are effective therapeutic strategies to improve both metabolic dysfunction and inflammatory status resulting in an improved glucose tolerance. However, the identification of definitive molecular targets for both training regimes, which seem to address slightly different signaling pathways, has to be elucidated in future studies.

Conflict of Interests

The authors declare no conflict of interests.

Authors' Contribution

Cornelia Mardare contributed to data analysis and developed and wrote the paper. Karsten Krüger developed the experimental design, contributed to data generation, and reviewed the paper. Gerhard Liebisch conducted the ceramide analysis and contributed to paper writing. Michael Seimetz conducted the mRNA analysis from adipose tissue and contributed to paper writing. Aline Couturier and Robert Ringseis analyzed the mRNA expression in liver tissue and contributed to paper writing. Jochen Wilhelm contributed to data analysis. Norbert Weissmann and Klaus Eder commented on the paper. Frank-Christoph Mooren reviewed the paper. Cornelia Mardare and Karsten Krüger contributed equally to this work.

References

9. Oliveira A. G., Carvalho B. M., Tobar N., et al. Physical exercise reduces circulating lipopolysaccharide and TLR4 activation and improves insulin signaling in tissues of DIO rats. [ PMC free article] Retracted

10. Kawanishi N., Yano H., Yokogawa Y., Suzuki K. Exercise training inhibits inflammation in adipose tissue via both suppression of macrophage infiltration and acceleration of phenotypic switching from M1 to M2 macrophages in high-fat-diet-induced obese mice.

12. Abd El-Kader S. M. Aerobic versus resistance exercise training in modulation of insulin resistance, adipocytokines and inflammatory cytokine levels in obese type 2 diabetic patients.

32. Liebisch G., Drobnik W., Reil M., et al. Quantitative measurement of different ceramide species from crude cellular extracts by electrospray ionization tandem mass spectrometry (ESI-MS/MS)

50. Uysal K. T., Wiesbrock S. M., Marino M. W., Hotamisligil G. S. Protection from obesity-induced insulin resistance in mice lacking TNF- α function.

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Endurance and Resistance Training Affect High Fat Diet-Induced Increase of Ceramides, Inflammasome Expression, and Systemic Inflammation in Mice

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Hydrating Vs Moisturizing Skin Care | Bee Naturals - Natural Skin and Body Care Products

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Many see "hydration" and "moisture" as interchangeable words that describe the same thing, believing a skin hydrator is the same as a moisturizer. It is a common misconception, and an easy mistake to make. In reality, however, these are two very different types of products specifically formulated to serve a different purpose for the skin.