Effect of carbohydrate type on brain composition and senescence in aging, hyperinsulinemia prone obese LA/Ntul//-cp rats

Orien L Tulp^*
*Correspondence: Orien L Tulp, Department of Medicine, University of Science, Arts and Technology, Olveston, Montserrat, Email:

Received: 29-May-2023, Manuscript No. AJIM-23-100477; Editor assigned: 01-May-2023, Pre QC No. AJIM-23-100477 (PQ); Reviewed: 15-May-2023, QC No. AJIM-23-100477; Revised: 16-Jun-2023, Manuscript No. AJIM-23-100477 (R); Published: 14-Jul-2023, DOI: -

Keywords

Hypertension, drug adherence, generic, prescriptions, substitution, Nigeria

Introduction

Overview

Since the revision of the traditional food group’s pyramid several decades ago, much of the dietary lipid previously present has been replaced with carbohydrate sources, often in the form of sucrose in order to retain a satisfactory magnitude and delivery of palatability considerations for many commercially processed foods. The consumption of simple vs Complex Carbohydrate (CHO) sources exerts significant differences on glycemic parameters in man and animals, with the simple CHO of sucrose resulting in a greater magnitude of Hyperinsulinemia (HI) and glucose Intolerance (IGT) in obesity than in non-obese subjects. Inflammatory cytokines originating and residing in adipose tissue of obesity have been reported to contribute to DNA damage in neuronal and other tissues, impede cell replication and accelerate neural cell senescence. In the present study carbohydrate rich diets containing cornstarch or sucrose were fed to congenic lean and obese, non-diabetic rats until late adulthood at 10.5 months of age, resulting in decreases in brain DNA, protein, and total brain mass, with the greatest decreases observed in the hyperinsulinemia prone, sucrose fed obese animals, while brain mass and composition was normal in lean littermates consuming the same dietary regimen. The prevalence of obesity is now a serious concern among Westernized societies, where it is rapidly approaching epidemic proportions. Moreover, the prevalence of obesity and overweight conditions now represents one of the most challenging issues facing the delivery of health care, in large part due to its close association with comorbidities of hypertension and noninsulin dependent diabetes. In addition, chronic obesity and nutritional factors have been linked to premature dementia including Alzheimer’s disease in addition to impaired neurodevelopment and DNA damage in multiple tissues when it occurs during earlier, formative life stages of growth and development and in aging. The syndrome of insulin resistance, glucose intolerance, and chronic inflammation typically accompanies obesity in man and animals, often resultingin increased comorbidities and decreased life span even in the absence of the pathophysiologic impacts of hypertension or diabetes when the condition is left untreated. The mechanisms contributing to chronic insulin resistance have been attributed to multiple factors, including overnutrition, chronic hyperphagia and other dietary macronutrient imbalances, disordered glucocorticoid metabolism and actions, impaired cellular translocation of insulindependent GLUT4 transporters, in addition to immune dysfunctions. Macrophage infiltration of adipose tissue, often resulting in disordered immune functions is one of the earmarks of obesity-mediated immune dysfunction.

Among the immunologic dysfunctions, adipose tissue tends to attract macrophages, which lead to secretion of inflammatory cytokines including IL-6 and others, that once released may now circulate systemically and impinge on numerous central and peripheral tissues, where they may initiate pathophysiologic sequela including vascular lesions and likely microglial contributions to premature apoptosis of neuronal cells. In humans, the neuronal changes in obesity plus Alzheimer’s syndrome include shrinkage of brain volume and cognitive deficits that accompany the increased adiposity and brain shrinkage. Thus, the purpose of the current study was to determine key parameters of brain composition in older, congenic lean and hyper insulinemic obese male rats, and to correlate the changes in brain composition with the magnitude of adiposity [1].

Optimal nutrition has been shown to influence pre- and early postnatal growth and to be positively associated with multiple developmental parameters that may follow later in life in man and animals. In contrast, early malnutrition and undernutrition are often followed by reduced stature and other developmental parameters later in life, proportional to the magnitude of the nutritional depravation. Brain development can also be impacted by early nutritional factors, and in the most dire examples with reduced brain development and decreased brain DNA content. In epigenetic rodent obesity, however, pre- and early postnatal nutrition are presumed to be comparable to similarly reared lean littermates in that they typically share the same lactation, equal access to solid food and nutritional experiences by weaning and thereafter when fed and housed as littermates under standard laboratory conditions. Thus, deficits in a congenic animal model are unlikely to be associated with frank inadequacies in nutritional deficits, but may be associated with the hyperphagia and resulting metabolic sequela that accompany the epigenetic expression and subsequent development of hyperinsulinemia, typically followed by progressive increases in adiposity and obesity [2]. The epigenetic expression of obesity in the LA/Ntul//-cp (corpulent) rat strain occurs as the result of an autosomal recessive trait, and results in 25% of the offspring of heterozygous breeding pairs demonstrating early stigmata of the progressive development of obesity by 5 to 6 weeks of age.

The obese phenotype demonstrate hyperphagia, hyperinsulinemia, hyperamylinemia, hyperlipidemia including hypertriglyceridemia and impaired glycemic responses to a glucose tolerance within a few weeks of postweaning life. In this strain, however, the obese littermates develop impaired glycemic responses typical of peripheral insulin resistance, but to date have remained nondiabetic throughout their lifespan. The animals have remained Specific Pathogen Free (SPF) in an isolated colony throughout many generations in our laboratory. Thus, the primary aspect of metabolism that remains in the obese phenotype is the chronic, lifelong hyperinsulinemia and hyperamylinemia and their pathophysiologic sequel [3].

Materials and Methods

Animals were housed in large plexiglass cages lined with one inch of pine shavings, maintained at 22°C and 50% RH under standard housing conditions in littermate pairs. The only known difference between the lean and obese phenotypes was the epigenetic expression of the -cp trait for obesity, originally obtained from the Koletsky rat 18 and backcrossed into the longevity-prone NIH LA/N background strain for 12 cycles by Hansen to establish the congenic designation at the NIH. Rats were maintained on Purina chow (#5012) and free access to house water from weaning. At 6 weeks of age rats were placed on a nutritionally adequate diet containing 54% carbohydrate as a lower glycemic index diet containing cooked cornstarch, 20% protein, and 16% mixed fat plus essential vitamins, minerals and fiber or a higher glycemic index diet containing 54% sucrose in place of the cornstarch, as described by Michaelis et al. At 10.5 months of age animals were sacrificed by rapid decapitation with a small animal guillotine after a brief fast and cervical blood was obtained for later analysis.

The brain tissues were dissected and weighed to the nearest mg and 50 mg-75 mg tissue aliquots of representative samples taken for proximate analysis. The residual carcasses including the remaining brain tissues were frozen, homogenized in a Waring blender, lyophilized and subjected to gravimetric analysis for determination of protein and lipid content. Measures of protein content of brain and residual carcass were obtained with the methods of Dole and Meinertz for lipid content and the classic methodology described by Lowry for protein content as described previously. The DNA analysis in brain tissue was determined by the method of Burton. Data were analyzed by standard statistical procedures. The study was approved by the institutional animal care and use committee [4].

Results

The results of final body weights are depicted in Figure 1 and indicate that by 10.5 months of age, the obese phenotype weighed more than twice the weights of their lean littermates, despite having been reared identically with respect to both diet and environment and with equal ad libitum access to the nutritionally sound diet. The body weights of rats of both phenotypes fed the SUC diet weighed more than their ST fed littermate companions. In Figure 2 carcass fat content is depicted and indicates that the total fat content of the obese were markedly greater than occurred in their lean counterparts, and that the SUC diet was associated with greater fat content than the ST diet in both phenotypes. Carcass protein content is depicted in Figure 3 indicates that the protein content of obese rats was greater than in their lean counterparts, with little additional increase with the SU diet [5].

The brain wet weights and the brain weight to body weight ratios are depicted in Figure 4 and indicate that the brain weights of the obese rats weighed significantly less than their lean littermates at 10.5 months of age (p<0.05). In addition, the ratio of brain weight to body weight was also decreased in the obese phenotype. The effects of the higher glycemic index sucrose diet tended to result is yet smaller absolute brain weights when fed the SUC vs. the ST diet and resulted in a further moderate trend toward decreases in the brain weight to body weight ratios that were mostly secondary to the greater fatness in the sucrose fed obese phenotype. There appears to be a mild diet trend effect on final brain weights, with ST fed animals of both phenotypes trending to weigh slightly more than was recorded for their ST fed littermates. In addition, the ratio of brain weight to final body weight of obese animals is depicted in Figure 3 and indicates that brain weights were also significantly less than were observed in their similarly reared lean littermates (p ≤ 0.01)[6].

Brain total protein and DNA content are depicted in Figure 5 and indicate that measures of net protein and DNA content per brain were both decreased in the obese phenotype. (p ≤ 0.05 for both total protein and DNA). The effects of diet are depicted to the right of each set of bars, and while suggestive of a further decrease in protein and DNA content, only the brain DNA content of the obese sucrose-fed obese animals were suggestive of a significant negative trend by Pages L test trend analysis. Thus, the decreases in brain content are generally proportional to the decreased brain weight observed in this study.

Brain lipid content is depicted in Figures 4 and 5 indicate that although the amount of lipid per brain was less in the obese phenotype, the percentage of lipid content in the brains were similar in both phenotypes, suggestive of a proportional decrease in overall brain composition rather than a decrease in any specific chemical component. Thus, although the brain lipid mass was lower in the obese than the lean phenotype, the results are consistent with and correspond to the smaller brain mass observed in those animals. When the net brain mass is compared to final body weight, the proportion of brain tissue lipid to total body weigh was significantly less in the obese than the lean phenotype, likely reflecting the substantially greater adiposity and body mass of the obese phenotype (Figures 6,7) [7].

Discussion

The results of this study indicate that at 10.5 months of age, total brain weight and absolute lipid, protein and DNA content were decreased in the obese phenotype, despite having been reared since birth with biological littermates via the same nutritional and environmental conditions with the exception of a low glycemic index starch vs a higher glycemic index sucrose diet.

The sucrose diet resulted in greater fat accretion and greater final body weights in the sucrose fed than the starch-fed animals of both phenotypes. Although the study did not monitor daily caloric intake, numerous previous reports have demonstrated hyperphagia and hyperinsulinemia to occur throughout much if not all of the lifespan in the obese phenotype of this strain, and where the typical duration of the lifespan of the obese phenotype is about one third shorter than its similarly reared and housed lean littermates [8].

Moreover, Michaelis et al. reported that the hyperinsulinemia of sucrose-fed rats remained greater than occurred in starch fed rats of this strain. Previously, brain size and cellularity have been shown to be decreased under conditions of severe malnutrition and in humans that develop Alzheimer’s disease, but only limited reports tend to identify obesity as an isolated contributor to premature brain shrinkage or brain dysfunction. The decreases in brain DNA content in the present study indicate decreased neuronal cellularity and presumably decreased cognitive functions as well and may accompany the decrease in cell number. Although the animals were not subjected to cognitive evaluation and the decreased brain size and cellularity could not be directly correlated with cognitive decline in the present study. They do however permit an assumption of decreased overall cognitive potential, and one where the presence of inflammatory cytokines common to obesity may be considered as a contributing factor in the neurologic decline. Of interest, the substitution of sucrose for starch in the diets resulted in a trend effect toward decreased DNA and protein content, and it seems likely that with a larger number of animals per treatment group or a longer duration of the dietary regimen that the diet induced results may have been more pronounced. In addition, since brain measurements were only undertaken at age 10.5 months, the chronologic timeline of the progression of neurologic decline or more profound effects of a high glycemic diet could not be firmly established in this study. Unlike dietary induced forms of obesity, where return to the normal diet may often be associated with weight loss, the obese phenotype in this epigenetic strain has to date proven to be remarkably refractory to significant weight loss or a return to the bodily habitus of their lean littermates following dietary intervention. To date, only excess daily T3 but not T4 administration has demonstrated significant increases in plasma T3, VO2 and in weight loss in the obese phenotype, with adrenalectomy resulting in partial but incomplete recovery [9].

The physiologic or metabolic basis for the differences in brain weight and composition are unclear but are highly suggestive of chronic hyperinsulinemia as contributing factors. Michaelis et al has previously reported impaired glucose tolerance and hyperinsulinemia in this strain from postweaning to 10 months of age.20 Thus, elevations in plasma insulin have been reported to occur through much if not the entire lifespan of the obese phenotype of this strain, and which contribute to the impaired thermogenesis in response to factors of diet and environment, including insulin inhibition of UCP1 actions in Brown Adipose Tissue (BAT). The BAT is a primary tissue in expressing nutritionally and environmental increases in thermogenesis, and when impaired due to metabolic aspects of insulin resistance in BAT and other peripheral tissues, results in increased rates of lipogenesis, metabolic efficiency and excess body fat accretion in both visceral and subcutaneous adipose tissue depots [10].

In lean animals, White Adipose Tissue (WAT) is enriched with type 2 immune cells, which interact with each other to generate type 2 cytokines including IL-4, IL-5 and IL- 13 to maintain a healthy type 2 immunologic protective physiologic environment. In contrast, with the development of obesity and in the presence of chronic hyperinsulinemia and may promote development of a type 1 inflammatory response, including the generation of inflammatory cytokines IL-6 and others which may produce damaging effects on neuronal DNA and neuronal survival. The type 1 inflammatory response includes metabolically activated macrophages, T-cells, B cells and others which release inflammatory cytokines, resulting in chronic systemic inflammation. The activated macrophages and other immune cells can collectively induce a type 1 inflammatory environment. The inflammatory cytokines can now migrate to virtually all somatic and neuronal tissues, where they may contribute to cellular senescence.

Dietary factors including excess fatty acids common to hypertriglyceridemia and obesity in concert with the lowgrade hypoxia that occurs in WAT have been shown to contribute to a chronic low-grade inflammation that can induce the pathophysiologic sequela including premature apoptosis among neuronal tissues, similar to that which appears to occur in Alzheimer’s disease [11]. The role of glucocorticoids in the development of obesity also merits some discussion [12]. Dysregulation of glucocorticoid actions may follow several lines of evidence. Glucocorticoids can induce both anti-inflammatory and inflammatory immune responses particularly from macrophages, which can generate both type 1 (secretes IL-6, inflammatory) and promote type 2(antiinflammatory) responses [13]. The physiologic response of glucocorticoids tend to be counterregulatory to those of insulin, creating a somewhat vicious cycle that may only become further aggravated in the presence of a chronic hyperinsulinemia state and which help to distinguish the impaired modulation of immune responses. Among other processes, dysregulation of glucocorticoid actions impede the formation and cellular translocation of GLUT4 glucose transporters from the endoplasmic reticulum of somatic cells, resulting in impaired cellular glucose uptake, thereby spiking further increases in insulin release mechanisms to insulin dependent tissues including skeletal muscle and adipose tissue, while promoting lipogenesis in liver and other receptive tissues.

Indeed a hallmark characteristic of genetically-obese rats if the progressive development of a fatty liver with advancing age, and which were visually apparent in all of the obese rats of the present study that were dissected [14]. Although glucocorticoids can induce cell death and reduce cell survival in immune cells such as T and B cells, macrophages in most tissues tend to be relatively resistant to glucocorticoid-induced apoptosis.

Overall, while glucocorticoids act to suppress systemic inflammation and are frequently prescribed to treat chronic inflammatory condition involving lymphocytes, they are less effective in macrophage-mediated diseases, such as chronic obstructive pulmonary disease and the chronic inflammation and hypoxia of obesity where an elevated release of inflammatory cytokines may occur differentially in different adipose tissue depots.

Regardless of the cellular processes implicated, the brain size, absolute composition, and cellularity based on DNA content was reduced in the aging adult obese non-diabetic phenotype of the LA/Ntul//-cp rat when fed a high glycemic index sucrose diet throughout much of their projected lifespan [15,16].

Conclusion

The results of the present study indicate that brain mass and cellular content at 10.5 months of age is decreased in the hyperinsulinemia-prone obese phenotype of this strain and are likely associated with a chronic inflammatory syndrome and cytokine expression that are common to hyperinsulinemia and obesity. Substitution of the higher glycemic index sucrose diet resulted in a trend toward further decreases in brain DNA and protein content, but the trends were not highly significant by conventional statistical analysis. The results could not determine the chronology of development the decrease or confirm an etiologic or developmental origin in brain size or composition as the data reflect only a single time point taken during late adulthood, in animals that typically often only survive for 12 to 15 months due to pathophysiologic complications of their obesity. In contrast the lean littermates have been observed to survive for 2 years or longer under similar environmental conditions, with obese females exhibiting a lesser magnitude of hyperinsulinemia and glucose intolerance and surviving longer than males.

The decreased brain size was characterized by proportionate decreases in total lipid, brain protein and brain DNA content, in association with marked body fat accretion, obesity and a decreased brain to body weight ratio in the epigenetic obese (-cp/-cp) phenotype of this congenic rat strain. The decreases in brain size and cellularity are also consistent with the brain shrinkage that occurs in Alzheimer’s disease and other states of dementia where chronic exposure to inflammatory cytokines may prevail and that occur in aging humans. The biological mechanisms or chronology through which the changes in brain composition occurred in the present study could not be determined from the single data point obtained, but were similar to the decreeses in brain mass and composition observed in obese animals fed a starch vs a sucrose diet reported elsewhere through much of their projected lifespan. Inflammatory cytokines of the macrophage-generated type 1 category include IL-6 and others and have been reported to generate free radical damage to DNA and contribute to neuronal senescence and thus remain an interesting causal speculation from the data obtained. Regardless of the Biological mechanism involved, the brain mass and apparent cellularity was significantly decreased in the obese phenotype of this rodent strain and the more highly refined high glycemic index sucrose diet tended to exaggerate the damaging impact on brain composition in the obese phenotypic of this strain.

Acknowledgment

The author thanks the late Dr. Otho E. Michaelis IV of the carbohydrate nutrition research laboratory, Beltsville MD human nutrition institute, Beltsville MD USA for the generous contribution of the experimental diet formulas used in this study and to Mr. Peisong Huang for his very capable assistance in animal husbandry and data collection.

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