Date: December 3rd, 2009

Speaker's Name and Affiliation:

• Deborah Clegg
• Touchstone Diabetes Center

Seminar Title: "Fatty-acid induced CNS Leptin and Insulin Resistance: A role for PKCs"

Chair: Harry R. Kissileff, Ph.D.

Rapporteur: Kathleen L. Keller, Ph.D.

Attendees and their Affiliation:

Kathleen Keller	Columbia/Obesity Research Center
Harry Kissileff	Obesity Research Center
TRhoda Gruen	Columbia
Tony Sclafani	Brooklyn College
Rich Bodnar	Queens College
Carol Maggio	NY ORC
Joseph Vasselli	NY ORC
Gerry Smith	Wiell Medical College
Ralph Norgren	Penn State College
Swana de Cysel	St. Luke's Roosevelt Hospital
Yann Ravussin	Columbia
Bridget Mueler	Columbia
Charles Mobbs	Mount Sinai
Katie Davis	UT Southwestern
JA Grinker	University of Michigan
Lori Zeltser	CUMC
Clemence Blouet	Einstein
Jill Carmody	CUMC
Angie Chong	CUMC

Summary: (Prepared by the Rapporteur)

Dr. Deborah Clegg of the Touchstone Diabetes Center presented an interesting seminar on the role of fatty acids in contributing to leptin and insulin resistance. Leptin and insulin are key adiposity signals and regulators of body weight homeostasis. But, these regulatory signals can be overridden by readily available sources of palatable and energy dense foods. Bray et al. and others have published epidemiological studies suggesting that dietary fat consumption is strongly associated with obesity. Dr. Clegg's work suggests this relationship might be due to more than just the high energy density of fat, compared to protein and carbohydrates. She hypothesized that exposure to a high-fat diet induces hypothalamic insulin/leptin resistance and she set out to test this hypothesis in a series of elegant experiments done in rats. Results demonstrated that as little as 72 hours of exposure on a high-fat (high in saturated fat/palmitic acid) diet cause hypothalamic insulin resistance. Further experiments concluded that exposure to a high-fat diet, regardless of adiposity, renders an animal insensitive to the anorexic effects of central insulin and leptin.

The next hypothesis explored was that exposure to a high-fat diet induces central insulin resistance by decreasing insulin intracellular signaling. Hypothalamic insulin signaling involves multiple steps, including increased phosphorylation of AKT (pAKT), and intraceullar mediator of insulin/leptin activity. Exposure to a high-fat diet decreases levels of pAKT, suggesting an interruption of intracellular insulin signaling. The next experiments addressed whether there were differences in this effect due to saturated vs. unsaturated fats. Dietary exposure to saturated fats caused hypothalamic insulin and leptin resistance and reduced insulin's ability regulate food intake/body weight and to impair hepatic glucose production. However, oleic acid, an unsaturated fatty acid found in olive oil, did not cause insulin and leptin resistance.

PKCs are enzymes that phosphorylate serine and threonine residues on many target proteins. At present, 10 PKC isoforms have been identified. Activation of PKCs involves translocation into the cystosol to binding domains at cell membranes. DAG facilitates the penetration of some PKCs into the cell membrane. Dr. Clegg has been investigating the role of PKCs, particularly PKC , in contributing to the insulin/leptin resistance caused by a high-fat diet. The first step, however, is locating PKC in the hypothalamus. Benoit, Kemp, and Clegg (JCI 2009) published via in situ hybridization and immunohistochemistry that PKC is expressed in the hypothalamus, specifically in the arcuate nucleus. Further data suggested that PKC was co-localized with NPY and leptin receptors, but not POMC, in the arcuate nucleus, suggesting it plays an important role in regulation of energy intake. To further establish a role for PKC , exposure to palmitic acid (saturated fat) diets induced translocation of PKC , but oleic acid had no such effect. Further, palmitic acid had no effect on other isoforms of PKC, suggesting its role might be specific for PKC (Benoit, Kemp, and Clegg JCI, 2009). Palmitic acid diets also caused reductions in hypothalamic pAKTand increased serine phosphorylation of IRS, both steps involved in the hypothalamic insulin/leptin resistance.

The next hypothesis explored by Dr. Clegg was that arcuate nucleus specific knock down of PKC attenuates diet induced obesity. Results demonstrated that in PKC knock down animals, intake of a high-fat diet was decreased. Also in these animals, glucose homeostasis and insulin induced pAKT was improved. Future experiments in Dr. Clegg's lab will investigate the role of other PKCs in the regulation of body weight homeostasis.

Question & Answers:

Q. What is the time course of the exposure? Exposure typically means that you taste something. Is that what you are referring to?
A. Initially animals are on a 3-4 week exposure to a high-fat diet and they got obese on this schedule. The animals had ad libitum consumption of this diet. Subsequent experiments employed a gavage technique of delivery which alleviated the taste/hedonic properties of the fatty acids.

Q. Were these animals pair fed or did you control the weight gain?
A. They were pair-fed.

Q. Can you get reversal of the hypothalamic insulin/leptin resistance that results due to high-fat diet exposure?
A. That's a great question. We're in the process of confirming this, but preliminary data suggest after return to a low fat diet for two weeks we were able to reverse the insulin/leptin resistance.

Q. This increase in brain palmitic acid resulted from 1 month on a high-fat diet?
A. Yes. We've also seen this response from shorter times on a high-fat diet, and specifically found that after only 3-days we see significant changes in the fatty acid content of the brain that mirrors the type of fatty acid the animals were exposed to.

Q. Is the oleic acid that you are using bound to albumin?
A. Yes. It's mixed with casein and albumin. The animals had no signs of being sick, so we think the diet was well-tolerated and that cannot explain our experimental effects. We tried bound and unbound fatty acids and the data were exactly the same

Q. Prior to the fatty acid, were the animals eating the same diet?
A. NO. The animals were maintained on the low fat diet ¨C and gavaged or infused the fatty acid and maintained on the chow diet.

Q. How many days after exposure to fatty acid does this effect occur?
A. As soon as 3 days after administration of fatty acid we will see that the animal shows signs of hypothalamic insulin/leptin resistance.

Q. Have you tried looking at the effect of glyceride esters of these fatty acids, since we do not eat fatty acids in large quantities in the diet.
A. We haven't tried that yet.

Q. What kind of fat was used in Shulman's work?
A. He used intra-lipid with a range of fatty acids.

Q. Why did you choose diacylglycerols instead of mono- or triacylglycerols?
A. Diacylglycerols are known activators of PKC

Q. Are you using adult animals?
A. Yes. I believe that the neonate primarily relies on ketone bodies for energy and thus the expression of PKC might not be as relevant with respect to CNS insulin/leptin sensitivity and fatty acdis.

Q. Why is the saline group lower in that figure?
A. I'm not sure that I've noticed that before. It's a good question. That saline animal is on a high fat diet ¨C it is possible that the presence of the fatty acids even in the basal state reduces pAKT.

Q. What is the role of PKC in peripheral insulin resistance?
A. PKC improves insulin signaling peripherally as well, so it is an important player in the development of insulin resistance.

Q. When you attenuated the ARC nucleus, did intake change?
A. Yes.

Q. What form of oil did you use in this study? Does it pass the blood brain barrier?
A. We used an emulsion. Given the data that the fatty acid composition of the brain changes to mirror that of the fatty acid given, we believe it does pass the blood brain barrier, although we are not sure how.

Q. Do we know if the fatty acid is actually getting into the brain, or is this insulin resistance occurring due to a similar mechanism as peripheral insulin resistance?
A. We tired to separate peripheral insulin resistance from hypothalamic insulin resistance by directly infusing the fatty acids into the brain and found that this changed leptin/insulin signaling as well. Our data are supportive of the fact that the fatty acids get into the brain, we are unsure of the mechanism.

Q. Have you tried other fatty acids besides palmitic acid?
A. We've tried DHA. In small doses, it is supposed to be anti-inflammatory, but in large doses, it is pro-inflammatory. In using the low doses of DHA we found that it did not induce insulin/leptin resistance. We are also thinking of trying mystiric and other saturated fatty acids but those are future experiments.

Q. On the molecular level with respect to insulin signaling, do you have any idea why saturated and unsaturated fatty acids would act differently?
A. I don't have a good answer for you. It might change the responsiveness of some of the cellular membranes. I'd love to know the answer.

Q. Do some of these molecular markers return to baseline after taking animals off the high-fat diet?
A. Yes, they do.

Q. Is the insulin you are measuring pancreatic in origin or brain in origin?
A. Probably pancreatic. It is highly controversial if insulin is made in the brain.

Q. Can you dissociate the weight gain effects from the nutrient/overfeeding effects by feeding animals on a lower-fat diet?
A. That's a good question. But how would you do that? By force feeding them?