BMAL1 Overexpression in Suprachiasmatic Nucleus Protects from Retinal Neurovascular Deficits in Diabetes

BMAL1 Overexpression in Suprachiasmatic Nucleus Protects from Retinal Neurovascular Deficits in Diabetes | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results BMAL1 Overexpression in Suprachiasmatic Nucleus Protects from Retinal Neurovascular Deficits in Diabetes Neha Mahajan , Qianyi Luo , Jodi Lukkes , Surabhi D Abhyankar , View ORCID Profile Ashay D Bhatwadekar doi: https://doi.org/10.1101/2025.02.05.636648 Neha Mahajan 1 Department of Biochemistry and Molecular Biology, Indiana University School of Medicine , 635 Barnhill Drive, Indianapolis, IN 46202 2 Indiana University, Stark Neurosciences Research Institute , 320 W 15 th St, Indianapolis, IN 46202 3 Department of Ophthalmology, Indiana University School of Medicine , 1160 W Michigan St, Indianapolis, IN 46202 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Qianyi Luo 1 Department of Biochemistry and Molecular Biology, Indiana University School of Medicine , 635 Barnhill Drive, Indianapolis, IN 46202 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jodi Lukkes 3 Department of Ophthalmology, Indiana University School of Medicine , 1160 W Michigan St, Indianapolis, IN 46202 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Surabhi D Abhyankar 1 Department of Biochemistry and Molecular Biology, Indiana University School of Medicine , 635 Barnhill Drive, Indianapolis, IN 46202 2 Indiana University, Stark Neurosciences Research Institute , 320 W 15 th St, Indianapolis, IN 46202 3 Department of Ophthalmology, Indiana University School of Medicine , 1160 W Michigan St, Indianapolis, IN 46202 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ashay D Bhatwadekar 1 Department of Biochemistry and Molecular Biology, Indiana University School of Medicine , 635 Barnhill Drive, Indianapolis, IN 46202 2 Indiana University, Stark Neurosciences Research Institute , 320 W 15 th St, Indianapolis, IN 46202 3 Department of Ophthalmology, Indiana University School of Medicine , 1160 W Michigan St, Indianapolis, IN 46202 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ashay D Bhatwadekar For correspondence: abhatwad{at}iu.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract The suprachiasmatic nucleus (SCN) regulates circadian rhythms and influences physiological and behavioral processes. Disruptions in circadian rhythms (CRD) are observed in type 2 diabetes (T2D), and importantly, CRD acts as an independent risk factor for T2D and its associated complications. BMAL1, a circadian clock gene, is vital for sustaining an optimal circadian rhythm and physiological function. However, the therapeutic potential of BMAL1 overexpression in the SCN to rectify the neurovascular deficits of T2D has yet to be investigated. In this study, db/db mice, a well-established model of T2D exhibiting arrhythmic behavior and the complications of diabetes, were injected stereotaxically with AAV8-Bmal1 or a control virus in the SCN to evaluate the protective effects of correcting the central clock on neurovascular deficits. Given the complex neurovascular network and the eye’s unique accessibility as a transparent system, ocular complications were selected as a model to examine the neuronal functional, behavioral, and vascular benefits of correcting the central clock. BMAL1 overexpression normalized the circadian rhythms, as demonstrated by improvements in the free-running period. The retinal neuronal function improved on electroretinogram, along with optomotor behavior and visual acuity enhancements. Retinal vascular deficits were also significantly reduced. Notably, our approach helped decrease fat content in genetically predisposed obese animals. Since the SCN is known to regulate hepatic glucose production via sympathetic mechanisms, glycemic control, and pyruvate tolerance tests were conducted. Systemically, we observed improved glucose homeostasis in BMAL1-overexpressing mice alongside a substantial reduction in hepatic gluconeogenesis. BMAL1 overexpression lowered plasma norepinephrine and liver TH levels, indicating a protective regulation of adrenergic signaling. Thus, this study underscores the therapeutic potential of targeting circadian clock genes like BMAL1 in the SCN to alleviate metabolic and neurovascular deficits associated with T2D. Our research offers a compelling framework for integrating circadian rhythms into managing diabetes and its complications. Download figure Open in new tab Introduction In mammals, a master circadian pacemaker, the suprachiasmatic (SCN) region of the hypothalamus, coordinates the circadian clocks in peripheral tissues to regulate 24-hour rhythms of physiological functions [ 1 ]. Transcriptional and translational feedback loops drive these circadian oscillations at the cellular level. The transcription factor brain and muscle ARNT-like protein 1 ( BMAL1 ) interacts with circadian locomotor output cycles kaput ( CLOCK ) gene to form a heterodimer, eventually to mediate the transcription of downstream negative regulators Period ( Per ) and Cryptochrome ( Cry ) genes. The Per/Cry heterodimer inhibits the expression and transcriptional activity of BMAL1 / CLOCK genes [ 2 ]. The fine-tuning and strict regulation of this circadian feedback loop is critical for circadian rhythmicity in mammals. BMAL1 is the only non-redundant gene in the central circadian clock, elevating its significance in various circadian rhythm-focused studies [ 3 ]. Interestingly, several studies have linked the BMAL1 abnormal expression patterns to metabolic complications, including type 2 diabetes (T2D). BMAL1 deletion has been shown to promote T2D, obesity, lipogenesis, and pancreatic β cell impairment [ 4 – 6 ], and conversely, pancreatic β-cell-specific overexpression of BMAL1 was found to be protective against obesity-induced glucose intolerance, and mice-overexpressing BMAL1 in the same study had better glucose-stimulated insulin release [ 7 ]. This growing evidence reflects the significant potential of BMAL1 in chronic diabetes. With the increasing prevalence of diabetes worldwide, the associated microvascular complications are growing rapidly among young, working-age adults [ 8 , 9 ]. The eye is uniquely positioned to study neurovascular complications of diabetes due to the combination of a transparent window and an intricate network of retinal neurons and vasculature. Additionally, it exhibits the most common complication of diabetes, diabetic retinopathy. The mammalian retina also has an autonomous circadian clock system independent of the SCN [ 10 ]; previously, we reported that circadian clock disruption negatively affects visual function in mice [ 11 ]. Several studies have shown the adverse effects of BMAL1 deletion on retinal cone and rod cells, which could accelerate retinal microvascular and macrovascular injuries [ 12 – 14 ]. These findings strengthen our premise for using the eye as a model system to assess the therapeutic benefits of correcting the circadian clock. We employ a novel therapeutic strategy for correcting overall circadian rhythm by overexpressing clock gene BMAL1 centrally in the SCN via stereotaxic delivery. We provide evidence that BMAL1 overexpression improves neuronal function and reduces acellular capillaries, i.e., vascular deficits of diabetes, by correcting systemic glucose metabolism. Importantly, the beneficial effects of BMAL1 overexpression were associated with decreased hepatic gluconeogenesis and noradrenaline signaling pathways. These observations implicate the potential therapeutic targeting of circadian clock genes for diabetes and its complications. Research Design and Methods Animals The B6.BKS(D)-Lepr db /J (an animal model for type 2 diabetes; db/db) and Lepr db / + db/m (heterozygotes; db/m) mice [stock number 000697] were procured from the Jackson Laboratory (Bar Harbor, ME, USA) and housed in the animal care facility at Glick eye institute, Indiana University. All the animals were kept under normal physiological conditions (12-hour light/dark conditions), with free access to food and water ad libitum. All the experiments performed were per the Guiding Principles in the Care and Use of Animals (National Institutes of Health) and the Association for Research in Vision and Ophthalmology’s Statement for the Use of Animals in Ophthalmic and Vision Research. AAV- BMAL1 delivery to the suprachiasmatic nucleus (SCN) AAV- BMAL1 was designed and procured from the Ocular Gene Therapy Core at the University of Florida, Gainesville, FL. The BMAL1 was cloned with smCBA promoter for ubiquitous expression with a GFP tag and subsequently packaged in AAV-8 for delivery and expression as smCBA-mBmal1-P2A-GFP, the AAV8-smCBA-GFP was used as a control virus. We performed a stereotaxic delivery of the AAV- BMAL1 vector using coordinates of bregma -0.96, lateral 0.5 mm, and ventral 9 mm for SCN delivery in 8-week-old mice. To validate the SCN deliveries, three mice were sacrificed four weeks post-stereotaxic surgeries followed by sectioning and staining. After successfully validating the SCN deliveries ( Suppl Fig 1 ), the mice were divided into the following groups: 1) db/m + NV (control mice/ No virus), 2) db/m + Cont (db/m with AAV only), 3) db/m + BMAL (db/m with AAV- BMAL1 ), db/db + NV (diabetic control mice/ No virus), 2) db/db + Cont (db/db with AAV only), 3) db/db + BMAL (db/db with AAV- BMAL1 ). All the mice were maintained for six months post-injections for the following listed experiments. Echo MRI Live unanaesthetised mice are guided into a plastic tube designed to restrict their movement but not constrain them. The animals are then subjected to a 1-4 min imaging period in the gantry using a low energy (0.05T) electromagnetic field. Based on emitted T1 and T2 relaxation curves, lean mass, fat mass, free water, and total body weight are calculated using standard algorithms. Wheel Running Activity Wheel running activity was measured using our previously reported method [ 11 ]. All the animals were housed individually in running wheel cages in sound attenuated and ventilated isolation cabinets (Phenome Technologies, Chicago, IL, USA) for 14 weeks. Wheel running activity was recorded every minute for 10 weeks using Actimetrics (Actimetrics, Chicago, IL, USA) hardware and monitored and analyzed using Clocklab (Actimetrics, Chicago, IL, USA). The mice were exposed to 12H light and 12H dark for the first 14 days and subsequently kept in the constant dark to assess the free-running activity rhythms. The actogram data from 15 to 70 days were used to determine the period and quantify wheel running activity using Clocklab. The period was determined by periodogram analysis (L12:D12). Wheel running activity counts were determined by averaging the total activity during light and dark phases and were expressed for 24 hours to make them comparable between groups. Neuronal Funciton using Electroretinogram (ERG) The retinal function was measured in the animals employing dark-adapted/scotopic ERG (LKC Technologies, Inc, Gaithersburg, MD, USA). All the animals were dark-adapted for 24 hours before proceeding with the ERG recordings. The mice were anesthetized by administering an i.p. injection of ketamine (100 mg/kg) and xylazine (5 mg/kg). The pupils were dilated using a topical application of 1% tropicamide and 2.5% phenylephrine (Alcon laboratories). The eyes were kept moist using a 2.5% hypromellose ophthalmic demulcent solution/Gonak (Akorn). For the ERG recordings, the ground needle electrode was placed on the base of the tail, and the reference electrode was sub-dermally placed between the eyes. The gold loop electrodes (LKC Technologies, Inc, Gaithersburg, MD, USA) placed over the cornea were used to record ERG response. The stimulus flash intensities of 0.025, 0.25, and 2.5 cd.s/m 2 for scotopic conditions were presented in a UTAS ganzfeld illuminator (LKC Technologies). The values for a wave and b wave amplitudes and their implicit times were obtained from an inbuilt analysis tool by LKC Technologies. Optomotor Response Behavior (OMR) The spatial vision was quantified in the animals by detecting the spatial frequency threshold of optometer response behavior using an OptoMotry device (CerebralMechanics, Inc.). Tracking head movements in response to rotating sine wave gratings (100% contrast) were recorded in free moving mice. Spatial frequency was systematically increased in a staircase method until the animal did not respond, and the highest spatial frequency the animal could track was identified as the threshold. The threshold obtained for each eye was reported. Immunohistochemistry for Tyrosine Hydroxylase The liver samples were fixed using 4% PFA in PBS for 24 hours, followed by paraffin embedding. The sections were placed on charged slides and dried at 56°C overnight. Slides were subsequently deparaffinized in xylene and hydrated through descending grades of ethyl alcohol to distilled water, and placed in Tris Buffered Saline pH 7.4 (Scytek Labs, Logan, UT) for 5 minutes for pH adjustment. The sections were then subjected to enzyme-induced epitope retrieval in 0.03% Pronase E/TBS (Millipore Sigma/Scytek) in an incubator at 37°C for 10 minutes, followed by several rinses in distilled water. Before proceeding with blocking for non-specific proteins with rodent Block M (Biocare, Concord, CA) for 20 minutes, the sections were pre-treated with 3% hydrogen peroxide/methanol for 30 minutes at 25°C, rinsed with distilled water, and washed with TBST for 5 minutes, followed by micro-polymer staining performed at room temperature on the Biocare intelliPATH automated stainer. Lastly, the sections were incubated with primary antibody (rabbit Tyrosine Hydroxylase (Millipore Sigma, Temecula, CA)) at 1:150 in Normal Antibody Diluent (Scytek) for 1 hour, followed by rodent HRP Polymer (Biocare) incubated for 30 minutes. Reaction development utilized Romulin AEC (Biocare) for 5 minutes, counterstained in CATHE Hematoxylin diluted 1:10 for 1 minute, followed by air drying, dipping in xylene, and coverslipping with permanent mounting media. The slides were imaged at 20X under a fluorescent microscope (Zeiss AXIO Observer.A1 Inverted Fluorescence Microscope, Carl Zeiss MicroImaging GmbH). Vascular deficits Animals were euthanized, and the eyes were enucleated and fixed in 4% paraformaldehyde. A day before trypsin digestion, the retinas were isolated following the previously reported procedure [ 15 ]. The isolated retina was placed in 50 mL water for unfixing overnight. The individual retina was incubated in 3% trypsin at 37°C for 2 hours the next day. The trypsin-digested retina was placed in a Petri dish, and the internal limiting membrane was gently separated from the peripheral retina with fine forceps. Then, using Vannas scissors, the internal limiting membrane was isolated from an optic nerve. Subsequently, the neural retina was removed, and the isolated retinal vasculature was stained with periodic acid and Schiff’s base to assess acellular capillary numbers. Norepinephrine levels using ELISA Whole blood was collected in an EDTA-coated collection tube before sacrificing the animals. After 30 minutes, the blood samples were centrifuged at 2000 x g for 20 minutes. The clear supernatant or plasma was separated and stored at -80° C. Plasma norepinephrine levels were quantified using a commercially available norepinephrine ELISA kit [cat no. 3836; Novus Biologicals LLC, CO, USA] as per the manufacturer’s guidelines. Glucose Tolerance Test (GTT) The animals were fasted for 4 hours, and basal blood glucose levels were monitored using a commercially available glucometer (AlphaTrek2) to set up basal or zero-time blood glucose. After which, glucose solution (1g/kg/bw) was administered via the i.p. route. The blood glucose was then measured again at 10, 20, 30-, 60-, 90-, and 120 minutes post-glucose administration. Insulin Tolerance Test (ITT) The animals were fasted for 2 hours for ITT, and basal blood glucose levels were quantified as described above. An i.p. injection of 0.5 IU/kg insulin Humulin R U-100 was administered to the animals, followed by measurement of glucose levels as described in GTT. Pyruvate Tolerance Test (PTT) The animals were fasted for 16 hours before basal blood glucose measurements and sodium pyruvate (P5280 Millipore Sigma, US) prepared in sterile PBS @ 1g/kg/bw was given intra-peritoneally. The blood glucose levels were afterward quantified using a glucometer for 15, 30, 60, 90, and 120 minutes as described earlier. Statistical Significance All the data were expressed as Mean ± SEM. The data was analyzed on GraphPad Prism V.10.0.0 for Windows (San Diego, California; www.graphpad.com ) using either one-way ANOVA or Brown Forsythe and Welch’s ANOVA test. Data were considered statistically significant when the p-value was less than 0.05. Results 1. BMAL1 overexpression improved free-running periods in db/db mice After BMAL1 overexpression in the SCN of db/db mice, we first investigated the voluntary free wheel running activity ( Fig 1A ) . We observed that db/db mice had reduced total activity compared to the db/m mice (suppl Fig 2) , and there was no effect of BMAL1 overexpression. The free running periods were significantly delayed in the db/db mice and db/db mice with the control virus ( Fig 1B ) . Interestingly, db/db mice overexpressing BMAL1 had significantly improved free-running period ( Fig 1B ) , suggesting that BMAL1 overexpression corrects the endogenous circadian rhythms in db/db mice. Download figure Open in new tab Fig 1: SCN- BMAL1 overexpression improves free-running periods. (A) Representative double-plotted actograms of wheel running activity under 12 hr. light and dark (LD12:12) and constant dark (DD) conditions. (B) Bar graphs presenting the free-running period of the respective groups. N: db/m+NV-5; db/m+cont-8; db/m+ BMAL -8; db/db+NV-4; db/db+Cont-6; db/db+ BMAL -9. The data is presented as Mean ± SEM and analyzed using Brown Forsythe and Welch’s ANOVA test where different symbols signify the following: * vs db/m + NV, *=p<0.05, ** = p<0.01; # vs db/m + Cont, # p<0.05, ## p<0.01; $ vs db/m + BMAL , $ p<0.05, $$ p<0.01 and & vs db/db + Cont, p<0.05. 2. BMAL1 overexpression improved OMR in db/db mice As we observed an improvement in the endogenous circadian rhythms of db/db mice, we studied the beneficial effects of central BMAL1 overexpression optomotor behavior. Firstly, we studied OMR tracking to check whether BMAL1 influences visual performance. The spatial frequency threshold was significantly reduced in the db/db and db/db + Cont, which was significantly improved in the db/db mice overexpressing BMAL1 ( Fig 2A ) . The contrast sensitivity was significantly lower in the db/db mice irrespective of BMAL1 overexpression compared to the db/m groups ( Fig 2B ). Download figure Open in new tab Fig 2: SCN- BMAL1 overexpression improved visual acuity behavior. (A) db/db mice overexpressing Bmal1 showed a significant increase in the spatial frequency threshold measured using an optomotor reflex tracking System. (B) Contrast sensitivity remained unchanged with the overexpression of BMAL1 . N= db/m+NV-5; db/m+cont-6; db/m+ BMAL -6; db/db+NV-12; db/db+Cont-7; db/db+ BMAL -6. The data is presented as Mean ± SEM and analyzed using One-way ANOVA followed by Tukey’s post-hoc test, * vs db/m + NV, * = p<0.05, ** = p<0.01, *** = p<0.001; # vs db/m + Cont, # = P<0.05, ### = p<0.001; $ vs db/m + BMAL , $ = p<0.05, $$ = p<0.01; @@@ vs db/db + NV, p<0.001 and &&& vs db/db + Cont, p<0.001. 3. BMAL1 overexpression improved retinal function in db/db mice Further, we examined the effect of BMAL1 overexpression on retinal function using an electroretinogram (ERG). Mice were subjected to scotopic ERG to evaluate bipolar cells and photoreceptor cells’ activity. There was a significant reduction in the b-wave amplitude in the db/db and db/db + Cont group, which was improved under BMAL1 overexpression in db/db mice, suggesting a protective effect of BMAL1 overexpression on bipolar cells in db/db mice ( Fig 3A ). Similarly, there was also a reduction in the b-wave peak latency time under BMAL1 overexpressing db/db mice ( Fig 3B ) . The a-wave amplitude and peak latency time, reflecting rod cells’ activity, were also improved in db/db mice overexpressing BMAL1 , but the difference was significant only at the 2.5 log cd.s/m 2 flash intensity ( Fig 3C &D) . The ERG analysis suggested that BMAL1 overexpression helps improve retinal function by improving bipolar cells and rod photoreceptors’ functions. Download figure Open in new tab Fig 3: SCN- BMAL1 overexpression improved neuronal function. (A) Scotopic a-wave quantification ( B ) a-wave peak latency time (C) Scotopic b-wave quantification (D) b-wave implicit time quantification. N: db/m+NV-10; db/m+cont-18; db/m+ BMAL -14; db/db+NV-19; db/db+Cont-15; db/db+ BMAL -17. The data is presented as Mean ± SEM and analyzed using Brown Forsythe and Welch’s ANOVA test; *p<0.05, **p<0.01, **p<0.001. 4. BMAL1 overexpression improved the vascular deficits in db/db mice After examining the behavioral functional vision using OMR and retinal functions using ERG, we next examined the vascular phenotype ( Fig 4 ). The db/db mice and db/db + Cont showed a significant increase in the acellular capillaries compared to the db/m groups ( Fig 4A and 4B ). The BMAL1 overexpression showed a significant reduction in the number of acellular capillaries in db/db mice, suggesting a preventive effect on DR ( Fig 4B ). Download figure Open in new tab Fig 4: A decrease in the vascular deficits in SCN- BMAL1 overexpressed db/db mice. (A) Representative images of trypsin-digested retinas from the respective groups and red arrows showing the changes in acellular capillary numbers. (B) Bar chart showing the quantification for the same. N= 5, Magnification 20X and Scale Bar 100µM; The data is presented as Mean ± SEM and analyzed using Brown-Forsythe and Welch ANOVA test; * vs db/m + NV, *p<0.05, **p<0.01; # vs db/m + Cont, #p<0.05, ##p<0.01; $ vs db/m + BMAL , $ p<0.05, $$ p<0.01; @ vs db/db + NV, p<0.05 and & vs db/db + Cont, p<0.05. 5. BMAL1 overexpression improved glucose homeostasis and physiological parameters in db/db mice While circadian rhythms, neurovascular deficits, and behavioral responses were corrected using our therapeutic strategy of correcting the central clock, the systemic effects of BMAL1 overexpression were not assessed. Reported literature suggests that BMAL1 knock-out animals exhibit weight gain and glucose intolerance [ 16 , 17 ]. To investigate the peripheral beneficial outcomes of central BMAL1 overexpression, we first evaluated the body composition of mice using EchoMRI ( Fig 5A-C ) . The body weight was significantly higher in db/db mice, while the BMAL1 overexpression led to a decrease in body weight when compared to the dbdb-control group ( Fig 5A ), the difference remained statistically insignificant. Strikingly, the fat mass was reduced significantly in BMAL1 overexpressing db/db mice compared to db/db mice. There was no observable difference in lean mass percentage among all the groups ( Fig 5C ) . Download figure Open in new tab Fig 5: Effect of SCN- BMAL1 on anthropometry and glucose homeostasis. ( A ) Body weight ( B ) Fat mass and ( C ) Lean mass measured using Echo MRI. ( D ) GTT and its AUC in respective groups. N for A-C: db/m+NV-4; db/m+cont-8; db/m+ BMAL -5; db/db+NV-4; db/db+Cont-3; db/db+ BMAL -8. N for D: db/m+NV-5; db/m+cont-10; db/m+ BMAL -10; db/db+NV-12; db/db+Cont-4; db/db+ BMAL -8. The data is presented as Mean ± SEM and analyzed using Brown-Forsythe and Welch ANOVA test; * vs. db/m + NV, *p<0.05, **p<0.01, ***p<0.001; # vs. db/m + Cont, #p<0.05, ##p<0.01, ###p<0.001; $ vs db/m + BMAL , $ p<0.05, $$ p<0.01, $$$ p<0.001; @ vs db/db + NV, p<0.05 and & vs db/db + Cont, p<0.05. Next, with the help of GTT, the effect of BMAL1 overexpression on glucose homeostasis was evaluated. A higher AUC in db/db and db/db + Cont mice reflected an impaired glucose clearance, which was improved after BMAL1 overexpression in db/db mice ( Fig 5D ) . We also examined insulin sensitivity using ITT, which showed a reduction in blood glucose clearance in db/db mice, and BMAL1 overexpression was unable to improve insulin-dependent glucose homeostasis in db/db mice (Suppl Fig 3). 6. BMAL1 overexpression improved hepatic gluconeogenesis through norepinephrine signaling BMAL1 is a well-known regulator of the central circadian clock. From our GTT, we observed an improvement in glucose intolerance. However, the insulin-dependent glucose homeostasis remains unchanged. Alternatively, BMAL1 also regulates hepatic glucose metabolism [ 18 ]. To ascertain whether similar mechanisms play a role in our study’s beneficial effects of central BMAL1 overexpression, we first performed a PTT, a routinely used test for hepatic gluconeogenesis. We found an increase in hepatic glucose production/gluconeogenesis in db/db, and db/db + Cont mice, which was significantly reduced under BMAL1 overexpression ( Fig 6A ) , suggesting a direct impact of SCN BMAL1 on hepatic gluconeogenesis and glucose metabolism. Download figure Open in new tab Fig 6: Effect of SCN- BMAL1 overexpression on hepatic gluconeogenesis and sympathetic nervous system. ( A ) Pyruvate tolerance test (PTT) and its AUC in respective groups. ( B ) Plasma norepinephrine (NE) levels were quantified using ELISA. N for PTT: db/m+NV-5; db/m+cont-8; db/m+ BMAL -5; db/db+NV-6; db/db+Cont-2; db/db+ BMAL -8. N for NE measurement: db/m+NV-5; db/m+cont-7; db/m+ BMAL -8; db/db+NV-10; db/db+Cont-6; db/db+ BMAL -5. The data is presented as Mean ± SEM and analyzed using Brown Forsythe and Welsch’s ANOVA test; * vs db/m + NV, *p<0.05, **p<0.01,**p<0.001; # vs db/m + Cont, ##p<0.01, ###p<0.001; $$ vs db/m + BMAL1 , p<0.01; and & vs db/db + Cont, & p<0.05, && p<0.01. Interestingly, hyperglycemia and impaired glucose homeostasis can elevate the sympathetic drive, increasing the norepinephrine levels [ 19 ]. Reciprocally, literature also suggests that poorly controlled diabetes could lead to an increase in plasma norepinephrine levels [ 20 , 21 ]. We were intrigued to investigate if central overexpression of BMAL1 could work through the sympathetic drive. So, we quantified plasma norepinephrine levels to understand the mechanism behind BMAL1 peripheral protective effects on glucose homeostasis. The norepinephrine levels were significantly higher in db/db mice, and BMAL1 overexpression in db/db mice reduced the circulating norepinephrine levels ( Fig 6B ). Moreover, we found an upregulation for tyrosine hydroxylase (TH) in the liver sections of db/db and db/db+Cont groups ( Fig 7 ) . TH is a rate-limiting enzyme in the synthesis of norepinephrine, therefore affecting the adrenergic pathways [ 22 , 23 ]. The immunohistochemistry experiment revealed that db/db mice activated the sympathetic nervous system, which was significantly regulated in BMAL1 overexpressing db/db mice ( Fig 7 ). Download figure Open in new tab Figure 7: SCN- BMAL1 overexpression downregulates Tyrosine hydroxylase (TH) expression. Representative images of TH-stained liver sections. Magnification: 20X and Scale Bar 100µM. N= 5 In conclusion, our data suggested that the sympathetic nervous system was activated in db/db mice, resulting in hepatic gluconeogenesis stimulation, which led to dysregulation of glucose homeostasis. BMAL1 overexpression in db/db mice prevented all these complications. Discussion Disruptions in circadian rhythms are linked to various metabolic disorders like diabetes, yet there has been little exploration of therapeutic strategies aimed at the circadian clock. This study highlights the protective role of BMAL1 gene overexpression in the SCN, impacting circadian rhythms, neurovascular deficits, and systemic physiological aspects in T2D. Furthermore, our findings offer distinctive mechanistic insights, illustrating how BMAL1 overexpression in the SCN influences sympathetic activity and glucose intolerance, a relationship that hasn’t been previously investigated. In mammals, the circadian rhythms are regulated by a master circadian clock located in the SCN of the hypothalamus, which in turn governs the peripheral circadian clock of peripheral organs. Interestingly, recent research has shown the downregulation of hippocampal BMAL1 in streptozotocin/high-fat diet-induced diabetic mice [ 24 ]. To facilitate the improvement in the central circadian clock, we injected AAV- BMAL1 in the SCN of db/db mice. The overexpression of BMAL1 in db/db mice improved the free-running period. This observation is consistent with the previous studies where BMAL1 deletion in arginine vasopressin neurons (AVP) of the dorsal SCN region lengthened the free-running period in AVP- BMAL1 knock-out mice [ 25 ]. Research has shown that diabetic mice experience a reduction in the expression of clock genes [ 26 – 28 ], which contributes to circadian arrhythmicity observed in db/db mice and leads to disrupted glycemic control [ 11 , 29 , 30 ]. Our findings indicate that by overexpressing BMAL1 in the SCN of diabetic mice, we could correct the central circadian clock. This intervention halted the rise in acellular capillaries and vascular deficits, which are typically hallmarks of the complications of diabetic retinopathy. Also, the reduction in b-wave amplitude in db/db mice was improved after BMAL1 overexpression in the SCN of db/db mice. Indeed, conditional BMAL1 knock-out mice also display retinal deficits, explicitly affecting the circadian rhythmicity of ERG b-wave amplitude [ 31 ]. However, in our study, we do not expect BMAL1 overexpression in SCN to directly impact retinal BMAL1 and influence ocular parameters, though we cannot exclude the potential for improved retrograde signaling. This intriguing possibility warrants further investigation in future studies. One of our study’s notable findings is a decrease in hepatic glucose production as the mode of action, which was not studied earlier in this context. The liver is a principal organ for glucose storage, and disruption in liver functions has detrimental metabolic consequences. Previously, injecting the transneuronal pseudorabies virus into the liver resulted in retrograde labeling of CNS neurons. Notably, the localization of third-order neurons in the SCN illustrates anatomical pathways that enable the biological clock to influence autonomic input to the liver, emphasizing the direct effects of restoring the SCN clock on liver function [ 32 ]. Moreover, sympathetic activation stimulates hepatic glucose production through gluconeogenesis and glycogenolysis, contrary to the effects of parasympathetic activation, leading to a reduction in glucose production [ 33 , 34 ]. Intriguingly; we observed an increase in the plasma levels of norepinephrine, a sympathetic neurotransmitter in db/db mice, and BMAL1 overexpressing db/db mice had a significant reduction in NE levels, suggesting impaired gluconeogenesis and glucose homeostasis in db/db mice. Moreover, as we delve deeper into the sympathetic system and hepatic gluconeogenesis as a principal model of action for the SCN’s effects, we also analyze the hepatic expression of the tyrosine hydroxylase (TH) enzyme. TH is a rate-limiting enzyme for synthesizing catecholamines (epinephrine, norepinephrine, and dopamine), known to be expressed in the nerve fibers around the portal vein, bile duct, or hepatic arteries of the liver. Consistent with the reported literature for a higher expression of TH enzyme in the liver sections of obese animals [ 35 ], we found a similar trend in db/db mice. However, with the overexpression of BMAL1 in db/db mice, the expression of TH was significantly decreased. We speculate that a higher hepatic TH expression with a simultaneous increase in the plasma norepinephrine levels leads to impaired glucose metabolism in diabetic mice. The correction of the central circadian clock via overexpressing SCN- BMAL1 has profound effects against peripheral impaired glucose homeostasis. So far, published literature has shown that NE can influence rodent livers’ circadian rhythms and clock gene expression [ 36 ]. Although the central overexpression of BMAL1 enhanced glycemic control, it did not impact insulin-dependent glucose clearance. This lack of change may be due to the genetic background of db/db mice (which are deficient in leptin receptors), as BMAL1 may interact with leptin and its receptors to influence various metabolic pathways, including insulin sensitivity and weight gain. [ 37 ]. It’s also important to emphasize that overexpressing BMAL1 in the SCN reduced fat mass in genetically obese mice. With recent advancements in anti-diabetic treatments (such as GLP-1 agonists and SGLT2 inhibitors) targeting central mechanisms, our approach paves the way for future pharmaceutical developments. Furthermore, while the SCN is known to influence leptin directly [ 38 ] leptin receptor-resistant mice allowed us to exclude leptin-mediated mechanisms, concentrating primarily on the SCN-mediated sympathetic pathway. In conclusion, our study shows that therapies to correct circadian misalignment could prove advantageous in treating diabetes and its related complications. Moreover, we found that correcting the central clock protects sympathetic nervous system activation and glycemic control. Collectively, these findings present a novel direction for using circadian clock genes as therapeutic targets for diabetes and related complications. Authors Contributions NM and QL initiated and led the animal experiments and wrote, reviewed, and edited the manuscript. JL led the AAV experiments and edited the manuscript. QL, NM and SA obtained and analyzed data and edited the manuscript. AB edited and reviewed the manuscript. AM reviewed the manuscript. All authors have given final approval for the version to be published. Funding This work is supported by funding support from National Eye Institute grants R01EY027779, R01EY027779-S1, and R01EY032080 to AB, a Challenge grant from Research to Prevent Blindness (RPB) to the Department of Ophthalmology. Conflict of Interest AB is an ad hoc District Support Pharmacist at CVS Health/Aetna. The contents of this study do not reflect those of CVS Health/Aetna. NM, QL, JL and SA do not have any conflicts to declare. Acknowledgment We want to thank Dr. Charlie Dong for the helpful discussion on liver studies, Ms. Kara Orr and Lata Udari for their technical help with GTT and ITT studies, Dr. Amy S Porter, Michigan State University, for the experimental help with tyrosine hydroxylase staining, and Dr. W. 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Share BMAL1 Overexpression in Suprachiasmatic Nucleus Protects from Retinal Neurovascular Deficits in Diabetes Neha Mahajan , Qianyi Luo , Jodi Lukkes , Surabhi D Abhyankar , Ashay D Bhatwadekar bioRxiv 2025.02.05.636648; doi: https://doi.org/10.1101/2025.02.05.636648 Share This Article: Copy Citation Tools BMAL1 Overexpression in Suprachiasmatic Nucleus Protects from Retinal Neurovascular Deficits in Diabetes Neha Mahajan , Qianyi Luo , Jodi Lukkes , Surabhi D Abhyankar , Ashay D Bhatwadekar bioRxiv 2025.02.05.636648; doi: https://doi.org/10.1101/2025.02.05.636648 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Neuroscience Subject Areas All Articles Animal Behavior and Cognition (7624) Biochemistry (17650) Bioengineering (13871) Bioinformatics (41882) Biophysics (21424) Cancer Biology (18566) Cell Biology (25461) Clinical Trials (138) Developmental Biology (13365) Ecology (19867) Epidemiology (2067) Evolutionary Biology (24290) Genetics (15590) Genomics (22476) Immunology (17713) Microbiology (40331) Molecular Biology (17148) Neuroscience (88477) Paleontology (666) Pathology (2828) Pharmacology and Toxicology (4816) Physiology (7635) Plant Biology (15114) Scientific Communication and Education (2044) Synthetic Biology (4286) Systems Biology (9815) Zoology (2268)