Diabetes results from dysfunctional carbohydrate metabolism that is caused by a relative deficiency of insulin. It has become a major threat to human health, the prevalence of which is estimated to be 2.8% worldwide (171 million affected) and is predicted to rise to 4.4% (366 million) by 2030. Both type 1 and type 2 diabetes continue to represent major challenges to the health and health care systems of the Canada and other countries (Statistics Canada). Current therapies are helpful, especially for type 2 diabetes, however they remain inadequate in preventing the negative effects of the type 1 diabetes and metabolic syndrome on the cardiovascular system, cancer and other aging-associated co-morbidities. It has long been recognized that exercise is an excellent first line therapy for both diabetes and obesity; however, many patients, especially morbidly obese, are unable to exercise sufficiently for a variety of reasons. A huge challenge has been to "capture" some of the benefits of exercise in a manner that can be useful medically for the very broad range of diseases for which exercise appears to provide benefit.
We and others have shown that endurance exercise induces metabolic adaptations via activation of the transcriptional co-activator peroxisome proliferator-activated receptor γ coactivator-1 α (PGC-1α) (17, 26, 31). PGC-1α is the master regulator of mitochondrial metabolism (24), and biogenesis that has been touted as a potential therapeutic target for aging-associated diseases, including diabetes (18). Interestingly, mild over-expression of PGC-1α in skeletal muscle alone is known to be protective against sarcopenia, to attenuate inactivity-induced fiber atrophy, to ameliorate ALS pathology, to reduce systemic chronic inflammation, and to maintain systemic glucose and insulin homeostasis in aged mice (8, 13, 25, 37, 38).
Recently, we have shown that 5 months of forced-treadmill endurance exercise training promotes systemic mitochondrial biogenesis and bioenergetic capacity, reduces mitochondrial DNA (mtDNA) mutations, prevented aging-associated phenotypes (including sarcopenia, cardiomyopathy, brain atrophy, osteoporosis, etc.), and increased lifespan of polymerase gamma mutator mouse model of aging (PolG mice) (28). Pertinent to this patent application, we have observed that with pre-mature aging, PolG mice (much like aging humans) show a massive reduction in pancreatic β-cell mass – classical marker of diabetic pathology (Figure 1). Five months of endurance exercise mitigated this reduction in pancreatic β-cell mass (Figure 1). Using our exerkines screen that we have developed (patent pending – property of Exerkine Corporation), we identified novel human and mouse gene, C19orf80 and Gm6484, respectively, of unknown function as a potential exerkine that promotes pancreatic β-cell mass expansion. We term both of these genes – Betalin.
Human C19orf80 (chromosome 19 open reading frame 80; NCBI Reference Sequence: NM_018687.6; http://useast.ensembl.org/Homo_sapiens/Gene/Summary?g=ENSG00000130173;r=19:11348178-11352619) and mouse Gm6484 (predicted gene 6484; NCBI Reference Sequence: NM_001080940; http://useast.ensembl.org/Mus_musculus/Gene/Summary?g=ENSMUSG00000047822;r=9:21835510-21837346;t=ENSMUST00000058777) are show over 70% homology and are evolutionary conserved proteins (Figure 2 a-b) with no reported biological function. Additionally, both human and mouse betalin proteins have classical signal peptide which ensures extracellular systemic secretion upon its synthesis (Figure 2a).
Since PGC-1α over-expression recapitulates many of the benefits of endurance exercise. We further utilized muscle-specific PGC-1α over-expression mouse model to study the expression of betalin in liver, heart, and skeletal muscle (quadriceps), and white adipose (subcutaneous inguinal fat) and brown adipose tissue depots. Here we show that PGC-1α over-expression in the skeletal muscle of the transgenic mice (MCK-PGC-1α) increases betalin expression in liver and fat pads (Figure 3a). Additionally, an acute bout of endurance exercise increases betalin expression in liver and fat pads of C57Bl/6J wildtype mice (Figure 3b). Since betalin expression is highest in liver as assessed from both MCK-PGC-1α study and acute endurance exercise study in wildtype mice, we believe that betalin belongs to hepatokine subclass of exerkines.
Given that betalin is induced in the liver of muscle specific-PGC-1α transgenic mice and with exercise (Figure 2), and is a secretory protein, we decided to evaluate its role in mediating cross-talk between liver and pancreas to expand pancreatic β-cell mass. We utilized adenoviral over-expression system to assess betalin role. Over-expression of betalin in mouse liver by hydrodynamic tail vein injection of adenovirus carrying betalin gene (Ad-GM6484) stimulates pancreatic β-cell proliferation vs. mice injected with control adenovirus carrying GFP gene (Ad-GFP). Pancreatic sections are stained with anti-insulin antibody (marker of pancreatic β-cell) and anti-Ki67 antibody (marker of cellular proliferation), while DAPI is used to locate nuclei. The expansion in pancreatic β-cell mass is significantly up-regulated by betalin over-expression in liver. Together, these results convinced us that exercise-mediated benefits in improving systemic insulin and glucose metabolism are in part regulated by betalin-induced pancreatic β-cell proliferation. We are now in process to study the therapeutic role of betalin in Akita mouse model of type 1 diabetes and in high-fat fed mouse model of obesity and type 2 diabetes.
Collectively, we have shown that circulating betalin is increased with exercise in rodents, and over-expression of betalin in mice stimulates pancreatic β-cell proliferation. Together the findings from these studies provide important insights into the basic science of muscle and exercise as well as open new avenues for the rapid development of new therapeutics to target the type 1 and type 2 diabetes pandemic.
We continue to explore the molecular mechanisms by which this exerkine increases energy expenditure and improves abnormalities in glucose homeostasis. This work has exciting promise for the development of new treatments for metabolic diseases (including type 2 diabetes, type 1 diabetes, and obesity) and other disorders that are improved with exercise.
TARGET POPULATION
The epidemic emergence of modern chronic diseases largely stems from the adoption of a sedentary lifestyle and excess energy intake (11). There is incontrovertible evidence from short-term and longitudinal epidemiological studies that endurance exercise extends life expectancy and reduces the risk of chronic diseases (1-7, 9, 10, 12, 14, 15, 19-23, 29, 30, 32-36). In fact, endurance exercise is the potent therapy against metabolic disturbances that lead to type 1 and type 2 diabetes. Despite this knowledge, type 1 and type 2 diabetes has reached pandemic state and there is an inflation in morbidly obese and diabetic adolescents, young and older adults. A great proportion of this population are unwilling or unable to exercise train. Hence, biomolecules like betalin that partake in inducing metabolic adaptations in response to exercise have lucrative therapeutic potential. Additionally, there is an increased recognition that patients that have primary mitochondrial myopathies can develop type 2 diabetes. Hence, betalin will have direct clinical implications in this patient population.
PROPOSED PATENT
We propose the use of betalin (C19orf80, Gm6484 and all synonyms) as a therapy for all forms metabolic syndromes with impaired glucose tolerance resulting in obesity, type 2 diabetes, type 1 diabetes, and insulin resistance (and not just limited to the aforementioned disorders). The patent would cover the use of purified, recombinant protein to be given to patients via infusion, subcutaneous injection, microencapsulated beads (encapsulated cell bio-delivery), or skin patch 3 – 7 times a week to raise the plasma concentration of betalin. We suggest that we cover dosing of betalin to raise the baseline concentration by at least 10 % above resting values to mimic the “pulse” with exercise.
EXPERIMENTAL METHODS for PILOT RESULTS
Breeding of PolG mutator mouse and littermate wildtype mice
Heterozygous mice (C57Bl/6J, PolgA+/D257A) for the mitochondrial polymerase gamma knock-in mutation were a kind gift of Dr. Tomas A. Prolla, University of Wisconsin-Madison, USA (16). We generated homozygous knock-in mtDNA mutator mice (PolG; PolgAD257A/D257A) and littermate wildtype (WT; PolgA+/+) from heterozygous mice-derived colony maintained at the McMaster University Central Animal Facility as previously described (28). During breeding, all animals were housed three to five per cage in a 12-h light/dark cycle and were fed ad libitum (Harlan-Teklad 8640 22/5 rodent diet) after weaning. The presence of the polymerase gamma homozygous knock-in mutation was confirmed as previously described (16, 28).
Endurance exercise protocol for PolG mutator mice
Endurance exercise protocol and tissue harvesting was carried out as previously described using an independent cohort of mice (28). Briefly, at three months of age, mice were housed individually in micro-isolator cages in a temperature- and humidity- controlled room and maintained on a 12-h light-dark cycle with food and water ad libitum (27, 28). Equal numbers of PolG female and male mice were assigned to sedentary (PolG-SED) or forced-endurance (PolG-END) exercise groups (n = 10/group; ♀ = ♂). None of the mice had been previously subjected to a structured exercise regiment. One week of pre-training was allowed to acclimatize the PolG-END mice to the treadmill. PolG-END mice were subjected to forced treadmill exercise (Eco 3/6 treadmill; Columbus Instruments, Columbus, Ohio) three times per week at 15 m/min for 45 min for five months. A 5-min warm-up and cool-down at 8 m/min was also included. PolG mice were age- and sex- matched with sedentary littermate WT mice (n = 10; ♀ = ♂), which served as controls for the study to assess if endurance exercise intervention can molecularly bring PolG mice to normalcy. At eight months of age animals were euthanized, and tissues were collected for molecular analyses. The study was approved by the McMaster University Animal Research and Ethics Board under the global Animal Utilization Protocol # 12-03-09, and the experimental protocol strictly followed guidelines put forth by Canadian Council of Animal Care. At the end of the study, pancreas was harvested and stained with anti-insulin antibody (Abcam).
Breeding of MCK-PGC-1 and littermate wildtype mice
Muscle-specific PGC-1 transgenic (C57BL/6-Tg(Ckm-Ppargc1a)31Brsp/J) and littermate wild-type (C57BL/6) were bought from Jackson Laboratories. MCK- PGC-1 transgenic mice express mouse peroxisome proliferative activated receptor, gamma, coactivator 1 alpha under the direction of the mouse muscle creatine kinase promoter. The colony is maintained at the McMaster University Central Animal Facility. During breeding, all animals were housed three to five per cage in a 12-h light/dark cycle and were fed ad libitum (Harlan-Teklad 8640 22/5 rodent diet) after weaning. The presence of the transgene is confirmed using genotyping protocol indicated by the Jackson Laboratories.
Acute endurance exercise protocol for wildtype mice
Male C57Bl/6J mice, bred in an institutional central animal facility (McMaster University), were housed in micro-isolator cages in a temperature- and humidity- controlled room and maintained on a 12-h light-dark cycle with food and water ad libitum. At 4 months of age, mice (N=10/group) were randomly assigned to either sedentary (SED) or forced-acute endurance exercise post 1-hour (Acute END +1hr) or forced-acute endurance exercise post 3-hour (Acute END +3hr) groups ensuring that body mass was similar between groups. None of the mice had been previously subjected to a structured exercise regime. Mice in both exercise groups were subjected to an acute bout of treadmill (Eco 3/6 treadmill; Columbus Instruments, Columbus, Ohio) running at 15 m/min for 90 min. A 5-min warm-up and cool-down at 8 m/min was also included. All of the mice in END exercise group completed the 90 min trial and were visibly exhausted (i.e., mouse will sit at the lower end of the treadmill, on the shock bar, for .5 seconds). Mice in the SED group served as controls. One or three hours following the acute bout of exercise, mice liver, heart, fat pads (inguinal and brown adipose tissue), and skeletal muscle (quadriceps) were harvested. Our exercise studies Animal Utilization Protocol is approved by the McMaster University Animal Research and Ethics Board under the global Animal Utilization Protocol # 12-03-09, and the experimental protocol strictly followed guidelines put forth by Canadian Council of Animal Care.
Total RNA isolation and mRNA expression analyses
Total RNA was isolated from tissues (liver, heart, fat, and skeletal muscle) using the Qiagen total RNA isolation kit (Qiagen, Mississauga, ON.) according to the manufacturer’s instructions. RNA samples were treated with TURBO DNA-freeTM (Ambion Inc., Austin, TX) to remove DNA contamination. RNA integrity and concentration was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) (27). The average RIN (RNA integrity number) value for all samples was 9.2 ± 0.2 (scale 1 – 10), ensuring a high quality of isolated RNA. The mRNA expression of genes involved in metabolism and browning gene program were quantified using 7300 Real-time PCR System (Applied Biosystems Inc., Foster City, CA) and SYBR® Green chemistry (PerfeCTa SYBR® Green Supermix, ROX, Quanta BioSciences, Gaithersburg, MD) as previously described (27). First-strand cDNA synthesis from 500 ng of total RNA was performed with random primers using a high capacity cDNA reverse transcription kit (Applied Biosystems Inc., Foster City, CA) according to manufacturer's directions. Forward and reverse primers for the aforementioned genes were designed based on sequences available in GenBank using the online MIT Primer 3 designer software (developed at Whitehead Institute and Howard Hughes Medical Institute by Steve Rozen and Helen Skaletsky), and were confirmed for specificity using the basic local alignment search tool. β-2 microglobulin was used as a control house-keeping gene, as its expression was not affected with the experimental intervention. All samples were run in duplicate simultaneously with a negative control which contained no cDNA.
Immunohistochemistry
Mouse pancreas was fixed in 4% paraformaldehyde. Cryosections were pretreated with citrate buffer for antigen retrieval according to standard protocols and were immunostained with Guinea pig anti-insulin antibody (Dako) and rabbit anti-Ki67 antibody (Abcam). Images were obtained using Olympus IX51 microscope.
Construction of adenoviral vectors
The Gm6484 expression vector was amplified from liver cDNA. The expression vector was subcloned into the pENTR1a vector (Invitrogen) and recombined into the pAd-CMV-DEST-V5 vector (Invitrogen) and adenovirus was produced using the virapower system (Invitrogen), including three rounds of amplification. Thereafter, virus was concentrated using the Vivapure adenopack 100 (Sartorius Stedim Biotech) and buffer exchanges to saline reaching a concentration of 8–10.i.f.u.µl−1. A GFP-containing adenovirus was prepared in parallel.