Diabetes & The Endocannabinoid System: Prospects For Therapeutic Control
By:
Matthew Schnur
Quick Outline
•	This will be a very detailed discussion, so lets put it in perspective
•	First we’ll discuss causes of diabetes
•	Then move on to insulin receptor signaling and defects in this mechanism
•	Next we will focus on the PPARã and cannabinoid CB1 & CB2 receptors
•	Finally, it will all be tied together; how cannabinoid therapy treats the symptoms of Type 1 & Type 2 Diabetes

Diabetes Background
•	Over 28 million Americans have diabetes (Type 1 or 2)
•	80% of cases are diagnosed as Type 2
•	The leading cause of blindness and amputations
•	Diagnosed cases are rising exponentially-directly related to diet
•	For every kg bodyweight over healthy BMI, a 7% increase in getting Type 2 is found
What is Diabetes?
•	Type 1 (Diabetes Mellitus)

–	An autoimmune disorder characterized by islet â-cell destruction 

–	Plasma glucagon levels may be increased

–	No detectable plasma insulin
What Is Diabetes?
•	Type 2 (Diabetes Insipidus)
–	Often environmentally induced in predisposed individuals
–	Characterized by:
•	Obesity		
•	Impaired IRS phosphorylation
•	Impaired PI3K activity
•	Impaired GLUT-4 translocation
•	Increased FFA

Common Attributes To Both
•	Both Type 1 and 2 patients have;
–	Hypo/hyperglycemia
–	Dyslipidemia
–	Decreased immune function
–	Poor wound healing
–	Microangiopathies
•	Neuropathy, retinopathy, nephropathy
–	Depression & weight gain
•	Both attributable to inflamm. TNFá, IL-2, and IL-6
Causes of Diabetes
•	Type 1:
–	Only 30% identical twins will both have it
–	MHC genes on chromosome 6
•	Of 21 known DR alleles, DR3 & DR4 found in 95%
–	â-cell autoantibodies
•	Directed against GAD (glutamic acid decarboxylase), unique to â-cells
Causes of Diabetes
•	Type 2
–	A variety of theories, we’ll focus on PPAR based
–	Interruption of lipid homeostasis
-	Leads to increased FFA
-	FFAs normally decreased by PPAR activation
2.  Activation of inflammatory cytokines normally suppressed by PPAR
Insulin Receptor Signaling
1.  Insulin binds to the heterotetrameric IR (Insulin Receptor)
	- Causes autophosphorylation of tyrosine residues

2. Tyrosine autophosphorylation causes dissociation of IRS-1 (Insulin Receptor Substrate-1)
	- 4 IRS proteins;
		* IRS-1 – immediate activation 	       of PI3K
		* IRS-2 – prolonged activation of 	        PI3k
		* IRS-3 & -4 – inhibit PI3K 	  	        activation

Insulin Receptor Signaling
3.  Activation of PI3K
	- Responsible for:
	     * Activ. of Akt/PKB  (serine 	  	      phosphorylation)
	     * GLUT-4 translocation
4.  Activation of Ras/Raf
	- Both PKB mediated or directly IRS     	activated
	-Activates the MEK- ERK1/2 	pathway
5.  MEK & ERK1/2 Pathway
	- Responsible for glycolysis & 	protein synthesis
	- Activation of PPARã
Insulin Desensitization
•	Besides tyrosine autophosphorylation, the IR has;

	- Both serine & threonine residues capable of 	autophosphorylation

	- Upon excess agonist activity, serine/threonine autophosp. causes a dissociation of IRS-1 without activation

	- Results in loss of function IR, or only activation of IRS-2
		* This is why we see �ª IRS-2 activity in both Types

Insulin Desensitization
•	Increased Fatty Acids
- Elevated FFAs lead to accumulation of
	* DAG		*fatty acyl-CoA
	* ceramide
- These compounds are known to activate membrane bound PKCè
- PKCè causes serine phosphorylation of IRS-1 in lieu of IR mediated IRS-1 tyrosine phosphorylation
	* Serine phosphorylation causes a dissociation 	between IRS-1 & PI3K

Insulin Resistance
3.  TNFá and inflammatory adipokines
	- Chronic exposure to TNFá to 3T3-L1 adipocytes 	resulted in 90% �« in GLUT-4 mRNA
	- TNFá has been found to:
		* Repress expression of IRS-1 & GLUT-4
		* Induce serine phosphorylation of IRS-1
		* Increase FFA plasma levels
	- TNFá levels >2.5x higher in both Type 1 & 2 than in	healthy patients

PPARã
•	Peroxisome-proliferator activated gamma (PPARã)
•	A nuclear receptor when activated dimerizes with retinoic X receptor
•	A downstream mediator of IR – MEK- ERK1/2 pathway
•	Both PPARã & retinoic X receptor activation shown to enhance insulin sensitivity
•	Ligands include mono- & poly-unsaturated fatty acids, PGs, the most commonly prescribed Type 2 diabetes medications thiazodolines (TZDs), and  some NSAIDs (possible breakdown to AM404)
Functions of the PPARã
•	Originally discovered to inhibit lipid peroxidation
•	Agonist activity found to down regulate TNFá gene
•	Stimulates adipocyte differentiation & apoptosis
–	Beneficial mostly for Type 2
•	Represses gene expression of chemokines involved in 	insulin resistance:
•	Leptin			* Plasminogen activator-inhibitor-1
•	Resistin		* IL-6 & IL-11
•	Induces gene expression of insulin sensitizing factors:
•	Adiponectin		* Fatty acid transport protein
•	IRS-2
The Endocannabinoid System
•	The CB1 & CB2 receptors are the most abundant G-protein coupled receptors in the human body
•	Besides CB1 & CB2 endo- & phyto- cannabinoids also bind to the PPARã and TRPV1 vanilloid receptor
–	The vanilloid receptor is expressed both in the islet â-cells and smooth muscle cells
–	Vanilloid receptor activation found to enhance insulin secretion and sensitivity
•	Anandamide (arachidonylethanolamide) & 2-AG (arachidonylglycerol) are endocannabinoids
–	These are under negative control of leptin

Endocann. Continued
–	Leptin is a hormone secreted by adipose tissue and exerts its effects in the hypothalamus

–	As previously mentioned, leptin increases insulin resistance

–	Endocannabinoids are down-regulated by leptin
•	Leptin causes an inhibition in the MAPK stimulated glycogen synthase activity of the CB1 receptor

The Cannabinoid Receptors
•	The CB1 & CB2 receptors
–	Both GPCR with Gái/o coupling
–	CB1 also has Gás coupling ability under certain conditions
–	Both coupled to activation of the PI3k-Akt/PKB pathway
–	Both receptors shown to activate MAPKs via the Ras/Raf pathway
•	P38 & p42/p44 MAPKs activated
•	Shown to increase glycogen storage, glucose metabolism, c-fos expression
CB Receptors Continued
–	Both receptors found to activate PLC
•	PLC cleaves IP3
•	IP3 releases Ca2+ from intracellular storage vesicles
–	CB1 receptor also shown to inhibit K+ outflow & Ca2+ efflux
–	CB2 not coupled to ion channels
CB & IR Interactions
CB Agonists
•	Thus CB1 activation beneficial to insulin sensitivity and glucose metabolism
•	CB2 is found predominantly in immune cells & adipocytes
•	CB2 activation in B-cells, macrophages, T-cells, and monocytes is found to:
–	Reduce TNFá, IL-2, IL-6, and IL-11; all elevated in diabetics and correlated to insulin resistance
–	Balance Th1/Th2 inflammatory cell profile
•	Autoimmune Type 1 diabetes has �ª activation of TH1/TH2
•	IFN-ã, IL-12, and TNFá associated with �ª TH1, treatment with THC showed a marked decrease in mRNA levels of all
CB Receptors & â-Cells
•	Insulin secretion by â-cells follows an oscillatory pattern
–	Stimulated by �ª &�« pattern of intracellular Ca2+
•	Receptor localization:
–	CB1 found mostly on á-cells
–	CB2 found on both á- & â-cells
–	TRPV1 also found on â-cells
•	Cannabinoids found to/may:
–	Reduce insulin secretion (metabolic syndrome X)
–	CB1 may reduce cAMP dependent release of glucagon
–	Enhance effects of insulin signaling

CB Receptors & â-Cells
•	The Evidence:

–	Anandamide & 2-AG concentration in â-cells �ª under hyperglycemic conditions and decreases under hypoglycemic conditions

–	Administration of insulin �« endocannabinoid levels

–	Chronic activation of CB1 leads to up-regulation of PPARã (in adipocytes)

–	Personal data: 
•	Smoking + insulin = ~18%> reduction in BGL
•	Smoking alone = ~8% reduction
•	No reduction when large quantities cannabis used + food
•	Dangerous enhancement between exercise + cannabis + insulin combination can reduce insulin by 1/5

Non-CB Mediated Effects
•	Both endo- & phyto- cannabinoids bind to the PPARã receptor
•	Diabetics have a marked reduction in immune function & O2 transport
- IgA glycosylation 4x �ª in both types of diabetics w/o complications, 33% more in Type 1
- IgM glycosylation �ªeven in healthy diabetics, 8% more in Type1
- Healthy individuals have 1-3% hemoglobin glycosylation, uncontrolled diabetics 20% (diagnostic tool HbA1c)
- Poor O2 transport by Hb leads to microangiopathies
- Other long lived proteins also get glycosylated; collagen, albumin, myelin
Non-CBR Mediated Effects
–	Since protein glycosylation is an oxidative process, antioxidants have proven useful
•	Preventative effects of Cannabis derived antioxidants on Hb glcosylation at [.5], [5], and [10]ìg
–	Quercitan (flavanoid) 3%, 37%, 52%
–	Kaempferol (terpenoid) 10%, 12%, 15%
–	20 other flavanoids, also THC, CBD, CBC, and CBG all have antioxidant properties
–	Hb glycosylation a Fenton Reaction
•	NIH published paper on cyclic voltammetry & rat focal ischemia model:  THC 20X potent the antioxidant than ascorbate
3.  Cannabinoids (CBD) protect against myelin degradation, and excessive glutamatergic firing, a cause of one type of diabetic neuropathy (sensory)
	- NMDA receptor induced intracellular Ca2+ accumulations cause neurotoxicity

Diabetic Retinopathy
•	2 Phases:

- Nonproliferative
•	Neovascularization – resp. for
dev. of new blood vessels in
many tissues, especially the retina
•	Growth mediated by VEGF

-	Proliferative phase
•	Advanced stages of retinopathy
•	Neovasc. Causes optic nerve damage & macular edema
•	Leading cause of blindness
•	¾ all diabetics after 15 yrs
Retinopathy
•	The VEGF Pathway
–	Also actiavtes the PI3K-AKT/PKB pathway (like the CB receptors)
–	Also activates the Ras/Raf dep. MAPK pathway just like the CB receptors
–	Yet again, also activates the PLCã-PKC pathway, and IP3 mediated intracellular Ca2+ release, like the CB receptors
–	How then, can cannabinoids be beneficial?

Retinopathy & The CB Receptors
How Cannabinoids Benefit Retinopathy:
•	Remember, 20 flavanoids + cannabinoid are antioxidants
-	The eye is rich with FFAs which are subject to oxidation (COX-2), typically elevated in diabetics
-	Cannabinoids prevent superoxide anion formation, and increase fatty acid metabolism
-	VEGF
        -        While VEGFR2 & CB receptors share nearly identical transduction   	 	  mechanisms, cannabinoids inhibit VEGF gene transcription via other 	 	  receptors, may not share similar phosphorylation patterns
        -        TNFá increases VEGF mRNA, as does the Ils that are inhibited by CB 	  activation
-	PEDF
        -        Pigment epithelial derived factor, a potent inhibitor of neovascukaarization via 	  VEGF
        -        PEDF is inhibited by oxidative stress & TNFá

Conclusions
•	Diabetes is a simple disorder with complex pathways regulating insulin resistance/sensitivity and secondary pathology 
•	Nearly all complications to diabetes are the result of hyperglycemia
•	After reviewing the IR, PPARã, CB1, CB2, and VEGF, we find that cannabinoid therapy for diabetes can:
•	Reduce BGLs		2.  Reduce HbA1c
•	�ª insulin sensitivity	4.  �ª glucose & lipid metabolism
•	Prevent retinopathy	6.  Inhibit inflammatory chemokines
•	Neuroprotection	8. Improve O2 transport 
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